Molecular Diagnostic of Chicken Parvovirus (ChPV) Affecting Broiler Flocks in EcuadorABSTRACT
Enteric diseases affect poultry and cause important economic losses in many countries worldwide. Avian parvovirus has been linked to enteric conditions, such as malabsorption and runting-stunting syndrome (RSS), characterized by diarrhoea, and reduced weight gain and growth retardation. In 2013 and 2016, 79 samples were collected from different organs of chickens in Ecuador that exhibited signs of diarrhea and stunting syndrome, and analysed for the presence of chicken parvovirus (ChPV). The detection method of ChPV applied was Polymerase Chain Reaction (PCR), using primers designed from the conserved region of the viral genome that encodes the non-structural protein NS1. Out of the 79 samples, 50.6% (40/79) were positive for ChPV, and their nucleotide and amino acid sequences were analysed to determine their phylogenetic relationship with the sequences reported in the United States, Canada, China, South Korea, Croatia, Poland, Hungary, and Brazil. Strong similarity of nucleotide and amino acid sequences among all analyzed sequences and between the analysed and reference sequences was demonstrated, and the phylogenetic analysis clustered all the sequences within the same group, demonstrating a strong relation between the studied strains and the reference chicken parvovirus strains.
Keywords: Chicken parvovirus; enteric diseases; molecular diagnostic; PCR
INTRODUCTION
The intestinal health of birds is related to animal welfare and the productive capacity of animals. Enteric problems cause economic losses around the world, especially in young chickens, due to the costs of therapeutic treatments, decreased productivity and even increased morbidity and mortality. Viral diseases are characterized by the presence of diarrhoea, decreased weight gain, and increased feed conversion (Goodwin et al., 1993; Otto et al., 2006; Pantin-Jackwood et al., 2008; Kang et al., 2012). Several viruses are associated with enteric problems in chickens, such as avian coronavirus (IBV), avian reovirus (AReo), chicken astrovirus (CAstV), avian rotavirus-A (ARTv-A), fowl aviadenovirus (FAdV), and chicken parvovirus (ChPV) (Guy, 1998; Zsak et al., 2008; Nuñez & Ferreira, 2013), but there is limited information on the effects of individual viruses and their interactions on gut health (Pantin-Jackwood et al. 2008; Domanska-Blicharz et al., 2012; Mettifogo et al., 2014).
Avian parvovirus was first reported by Kisary et al. (1984),who found parvovirus-like virus particles that caused Derzsy’s disease in geese, using electron microscopy with gut samples from chickens with Runting-Stunting Syndrome (RSS).The family Parvoviridae contains two subfamilies: Parvovirinae that infect vertebrates, and Densovirinae that infect invertebrates (Nuñez & Ferreira, 2013).
The chicken parvovirus (ChPV)belongs to the genus Aveparvovirus, which also includes the turkey parvovirus (Cotmore et al., 2014). The particles of ChPV are small (19-24 nm in diameter), non-enveloped, and have icosahedral symmetry. The linear genome is single-stranded DNA and it is5 kilobases long (Kisary et al., 1984; Cotmore & Tattersall, 1995; Domanska-Blicharz et al., 2012).The genome contains 3 open reading frames (ORFs), including ORF 5’, which is 2085 nt long, ORF 3’,which is 2028 nt long, and a small ORF that is 306 nt long, located between 5’ and 3’ ORFs. The 5’ORF encodes a non-structural protein, NS1, whereas the 3’ORF appears to encode the capsid proteins VP1, VP2 and VP3, whereas the function of the small ORF has not been defined yet(Day & Zsak, 2010).
ChPV is related to enteric diseases that cause diarrhoea, growth retardation and lower than average weight gain, specially in 2- to 7-year-old chicks, and it is considered to be one of the aetiological agents for RSS (Zsak et al., 2013). This syndrome is also called malabsorption syndrome (MAS), helicopter disease, infectious stunting syndrome and brittle bone disease (Finkler et al., 2016).Viral replication and pathogenic effects mainly occur in cells with high proliferative rates (Hueffer & Parrish, 2003).
The aim of this study is to determine the presence of ChPV in organs obtained from broilers in Ecuador with signs of enteric disease, using Polymerase Chain Reaction (PCR) and nucleotide sequencing procedures.
MATERIALS AND METHODS
Samples
In 2013 and 2016, 79 samples were received at the Laboratory of Avian Diseases of the University of São Paulo, Brazil, corresponding to imprints of different organs, including the thymus, spleen, trachea, lung, air sac, gut, caecal tonsil, bursa, kidney and bone marrow of broilers between 1 to 4 weeks of age reared in Ecuador. Out of those samples, 42 were obtained in 2013, and 37 in 2016. The samples were used for the molecular analysis of enteric viruses that could be affecting commercial broiler flocks, whose clinical history included enteric problems such as diarrhoea, malabsorption, and delayed growth. These birds belonged to different commercial flocks distributed in the northern region of Ecuador, and after necropsy, several imprints were collected on FTA cards (GE Healthcare, Buckinghamshire, UK) for shipment to Brazil.
DNA Isolation
The material impregnated on the FTA cards was cut and suspended in PBS (Phosphate Buffered Solution), 0.1 M, pH 7.4, at 1:1 ratio, then macerated into 2-mLmicrotubes using a bead mill (TissueLyser LT Bead Mill, Qiagen, Hilden, Germany) for 5 minutes. The material was finally centrifuged for 30 min at 12,000 x g and at 4 °C. An aliquot of the supernatant was then collected for the extraction of DNA by the phenol/chloroform technique described by Chomczynski (1993). The extracted DNA was stored at -20 °C.
Polymerase chain reaction (PCR) for the detection of chicken parvovirus
The primers used in this reaction were those described by Zsak et al. (2009), PVF1 5’-TTCTAATAACGATATCACT-3’ and PVR1 5’-TTTGCGCTTGCGGTGAAGTCTGGCTCG-3’,corresponding to the conserved region of the non-structural NS gene, which amplify a 561-bp fragment. The PCR reaction conditions for ChPV amplification were performed as reported by Zsak et al. (2009), with some variations. PCR components were mixed in a DNA-free microfuge tube that included 1X reaction buffer, 1.25 mM of each deoxynucleotide triphosphate, 0.5 μM of each primer, 1.25 U of Platinum® Taq polymerase (Invitrogen® by Life Technologies, Carlsbad, CA, USA), and 2 μL of extracted DNA. Thermocycling parameters included one cycle of DNA denaturation at 94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by final extension at 72°C for 10 min. The PCR products of all samples were run on 1.5% Agarose gel using SyBR® Safe DNA gel stain (InvitrogenTM) and a 100 bp DNA Ladder (InvitrogenTM) to determine band size.
DNA sequencing and nucleotide sequence analysis
The amplified product was purified using the GPX™ PCR DNA and Gel Band Purification kit (GE Healthcare, Piscataway, New Jersey, USA), according to the manufacturer’s instructions. Each purified product was sequenced in the forward and reverse direction using the BigDye® Terminator Cycle Sequencing Kit v. 3.1 (Applied Biosystems by Life Technologies, Carlsbad, CA, USA). Sequencing reactions were carried out in ABI 3730 DNA Analyzer (Applied Biosystems by Life Technologies). The sequences obtained were edited using the CLC Main Workbench 7.7.3 software and aligned with previous reported sequences obtained from the GenBank database belonging to Brazil, Canada, Croatia, China, Hungary, South Korea, Poland, and the United States, using the CLUSTAL W method available in the ClustalX 2.1 software. Accession numbers of the reference sequences are detailed in the phylogenetic tree (Figure 1). The phylogenetic tree was inferred using the neighbour-joining method, with 1,000 bootstrap replicates integrated in the MEGA 7.0.18 software. The nucleotide and amino acid sequence similarity matrix was generated in the BioEdit Sequence Alignment Editor v. 7.2.5.
Phylogenetic analysis of the nucleotide sequences of ChPV from Ecuador. The sequence NC_001701.1 in red (goose parvovirus) was placed as a control outside the group. Numbers along the back refer to bootstrap values for 1,000 replicates. The scale bar represents the number of substitutions per site. The sequences obtained in the present work are shown in blue. EC=Ecuador, BR=Brazil, CA=Canada, HR=Croatia, HU=Hungary, PL=Poland, CH=China, US=United States, KR=South Korea.
RESULTS
PCR
PCR products were run on 1.5% agarose gel, and the location of the DNA band of each positive sample confirmed the amplification of the 561 bp segment in 40/79 samples, out of which 17/42 corresponded to the samples received in 2013 and 23/37 to the samples received in 2016. The details of the positive samples are described in Table 1.
Sample identification and origin, type of bird, clinical signs, year of collection and accession number from the NCBI GenBank database.
DNA sequencing and phylogenetic analysis
It was possible to sequence all positive results, obtaining a total of 40 sequences from different organs: 20 from bursae, seven in tracheas, three in caecal tonsils, two in spleens, kidneys, and thymuses, one in each of the following organs: air sac, bone marrow, intestine, and lung. The details of all positive samples, including GenBank accession numbers, are given in Table 1. The 40 sequenced fragments were analysed with a size of 398 nucleotides, showing a high percentage of similarity among nucleotides (NT)(89.6% - 100%) and amino acids (AA)(90.1% - 100%). Furthermore, there was a high percentage of similarity between sequences from Brazil (91.9% - 99.2% NT and 91.6% - 100% AA), Canada (87.9% - 94.2% NT and 87.8% - 96.2% AA), the United States (90.4% - 97.4% NT and 91.6% - 100% AA), Croatia (91.7% - 99.4% NT and 91.6% - 100% AA), Poland (92.2% - 98.2% NT and 91.6% - 100% AA), China (90.7% - 98.2% NT and 92.4% - 99.2% AA), South Korea (88.4% - 96.2% NT and 87.8% - 96.2% AA) and Hungary (91.9% - 98.7% NT and 92.4% - 99.2% AA). The similarity matrix is detailed in Table 2.
Matrix of similarity for nucleotide and amino acid sequences. To the left, nucleotide sequences, and to the top, amino acid sequences obtained in the study, compared with the reference sequences obtained from GenBank. EC=Ecuador, BR=Brazil (21), CA=Canada (22), HR=Croatia (23), HU=Hungary (24), PL=Poland (26 and 27), CH=China (29), US=United States (28), KR=South Korea (25). To the left, nucleotide sequences, and to the top, amino acid sequences obtained in the study, compared with the reference sequences obtained from GenBank. EC=Ecuador, BR=Brazil, CA=Canada, HR=Croatia, HU=Hungary, PL=Poland, CH=China, US=United States, KR=South Korea. (Part 1)
Matrix of similarity for nucleotide and amino acid sequences. To the left, nucleotide sequences, and to the top, amino acid sequences obtained in the study, compared with the reference sequences obtained from GenBank. EC=Ecuador, BR=Brazil (21), CA=Canada (22), HR=Croatia (23), HU=Hungary (24), PL=Poland (26 and 27), CH=China (29), US=United States (28), KR=South Korea (25). To the left, nucleotide sequences, and to the top, amino acid sequences obtained in the study, compared with the reference sequences obtained from GenBank. EC=Ecuador, BR=Brazil, CA=Canada, HR=Croatia, HU=Hungary, PL=Poland, CH=China, US=United States, KR=South Korea. (Part 2)
In the phylogenetic analysis, all sequences were clustered in the same group, demonstrating that the sequences obtained in this study are related to the reference sequences originating from North America, Brazil, Europe and Asia, as shown in Figure 1.
DISCUSSION
The primary aetiology of RSS or MAS in chickens is still unknown, although several viruses have been identified in birds with RSS, and ChPV being found in many of these disorders (Goodwin et al., 1993; Pantin-Jackwood et al., 2008; Domanska-Blicharz et al., 2012; Devaney et al., 2016). ChPV has a worldwide distribution, and it has been associated with enteric diseases in many other countries (Kisary et al., 1984; Decaesstecker et al., 1986; Goodwin et al., 1990; Zsak et al., 2008,2009;Bidin et al., 2011; Domanska-Blicharz et al., 2012;Tarasiuk et al., 2012;Nuñez et al., 2016). Experimentally, ChPV produces intestinal alterations such as diarrhoea, reduced weight gain and growth retardation (Zsak et al., 2013). In the present study, we searched for the presence of ChPV in different imprints of organs fixed in FTA cards collected from birds with enteric problems, such as diarrhoea and stunting. The results showed the presence of ChPV in 50.6% of the collected samples, demonstrating that the virus is not only related to enteric organs but also to organs of other systems, such as respiratory (trachea, lungs, and air sacs), immune (thymus, bursa, bone marrow and spleen), and urinary (kidney) organ, as previously demonstrated in the experimental studies of Zsak et al. (2013) and Domanska-Blicharz et al. (2012).
The parvovirus infections found in this study corresponded to young chickens, confirming previously published data on the occurrence of the virus in young animals (Palade et al., 2011; Domanska-Blicharz et al., 2012), which may indicate the occurrence of vertical infection in poultry farms in Ecuador.
In this study, we confirmed that the PCR protocol used for the amplification of a genome segment encoding the non-structural protein (NS1) in the 5’ORF region (Zsak et al., 2009) allowed for the identification of ChPV by the amplification of a 561-bp DNA fragment. Furthermore, we found a high percentage of similarity between the obtained nucleotide and amino acid sequences and others described and submitted to the GenBank from North America, Brazil, Europe and Asia. All samples used in this study derived from broilers affected with enteric disease, and therefore, it was not possible to determine the presence of ChPV in birds with no signs of enteric disease to corroborate the prevalence of natural infections of ChPV in healthy broiler flocks in the USA found by Zsak et al. (2008).
In conclusion, we confirmed the circulation of ChPV in poultry farms located in the northern region of Ecuador, providing the first molecular report of the virus in this country, which is possibly related to the enteric diseases described above. However, the exact role of the virus in enteropathiesis not fully understood, and thus, further pathological and epidemiological studies are needed to determine the real pathogenicity and prevalence of this pathogen in Ecuador, and to develop vaccines in the future to prevent the vertical and horizontal transmission of ChPV.
ACKNOWLEDGMENTS
The authors would like to the “Secretaría de Educación Superior, Ciencia, Tecnología e Innovación - SENESCYT” for its economic support through the Universities of Excellence 2014 scholarship programme of Ecuador. The authors would also like to thank the poultry companies in Brazil that generously sent the samples for the development of this study and for the diagnosis of enteric viruses. This work was supported by grants of FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) under#2013/08560-5 and 2015/09348-5, and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico) under #453920/2014-4 and 140744/2014-2.
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Publication Dates
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Publication in this collection
Oct-Dec 2018
History
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Received
11 Jan 2018 -
Accepted
03 Apr 2018
