Porcine parvoviruses in Polish pigs

ORIGINAL ARTICLE

Circulation of porcine parvoviruses types 1 through 6 in serum samples obtained from six commercial Polish pig farms

J. Cui1, K. Biernacka2, J. Fan1,3, P. F. Gerber1, T. Stadejek2 and T. Opriessnig1,4

1 The Roslin Institute and The Royal (Dick) School of Veterinary Studies, University of Edinburgh, Midlothian, Scotland, United Kingdom

2 Department of Pathology and Veterinary Diagnostics, Faculty of Veterinary Medicine, Warsaw University of Life Sciences, Warsaw, Poland

3 College of Veterinary Medicine, Agricultural University of Hebei, Baoding, China

4 Department of Veterinary Diagnostic and Production Animal Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa, USA

Correspondence:

T. Opriessnig; The Roslin Institute and R(D)SVS, University of Edinburgh

Easter Bush, Midlothian; EH25 9RG, Scotland, United Kingdom.

Tel: +44 (0)131 651-9422; E-mail: Tanja.Opriessnig@roslin.ed.ac.uk

Keywords:

Pigs; Porcine parvovirus (PPV); Prevalence; Poland.

Words in abstract: 271/300

Words in main text: 2604

Tables: 2

Figures: 2

Summary

Porcine parvoviruses are small non-enveloped DNA viruses, very resistant to inactivation, and ubiquitous in the global pig population. Porcine parvovirus type 1 (PPV1) has been known since the 1960s and is a major causative agent of reproductive failure in breeding herds. During the last decade, several new parvoviruses have been identified in pigs by molecular methods and have been consecutively designated as PPV2 through PPV6. Epidemiology data for these viruses is limited and the impact of these newly recognized parvoviruses on pigs is largely unknown. To further generate knowledge on the distribution of PPVs in pigs, a total of 247 serum samples were collected from six commercial Polish pig farms during 2013-2015 and tested by PCR assays and ELISAs. The pigs ranged from 2-18 weeks of age at sample collection. Breeding herds supplying the investigated farms were routinely vaccinated against PPV1. While all growing pig samples were negative for PPV1 DNA, young pigs were frequently negative for PPV1 antibodies and seroconversion to PPV1 was commonly seen at 9-10 weeks of age. The PPV2 antibodies detection was highest in young pigs (2 to 6-week-old) and decreased in older pigs indicating passively-acquired antibodies. The DNA prevalence rates in the serum samples analysed was 19% for PPV2, 7.7% for PPV3, 2.4% for PPV4, 4.0% for PPV5 and 6.1% for PPV6. Most PPV DNA positive samples were identified in 9 to 18-week-old pigs with no obvious association with disease on the farm. All recently emerging PPV genotypes were detected on Polish farms. Similar to previous reports in other pig populations, PPV2 was the most frequent PPV genotype circulating in Poland.

Introduction

Parvoviruses are small, non-enveloped DNA viruses with a single-stranded linear genome of approximately 4-6.3 kb in size (Xiao et al., 2013a). The family Parvoviridae has a broad host range and can be divided into two subfamilies: the Parvovirinae infecting vertebrates and the Densovirinae infecting arthropods. The subfamily Parvovirinae consists of eight genera of which Protoparvovirus, Bocaparvovirus, Copiparvovirus and Teraparovirus contain viruses that infect pigs (Streck et al., 2015).

Porcine parvovirus type 1 (PPV1) belongs to the genus Protoparvovirus and was first isolated in Germany in 1965 as a cell culture contaminant (Mayr et al., 1968; Mayr and Mahnel, 1964). PPV1 is considered one of the major causative agents responsible for reproductive failure in swine (Mengeling et al., 2000; Streck et al., 2013) causing economic losses in the swine industry worldwide (Mengeling et al., 2000).

During the last two decades, several novel parvoviruses have been identified in pigs using molecular methods and have been designated as PPV2 through PPV6. The PPV2 and PPV3 belong to the genus Tetraparvovirus. PPV2 was discovered in 2001 during a serum survey for hepatitis E virus in Myanmar (Hijikata et al., 2001). During 2006 and 2007, a similar virus called Cnvirus was identified in pigs co-infected with high pathogenic porcine respiratory and reproductive syndrome virus in China (Wang et al., 2010). Porcine parvovirus type 3 (PPV3), also known as porcine PARV4 and porcine hokovirus and closely related to human parvovirus 4 (PARV4), was identified in Hong Kong in 2008 (Lau et al., 2008). Three additional PPV genotypes, PPV4, PPV5 and PPV6, have been identified in recent years and belong to the genus Copiparvovirus (Streck et al., 2015). PPV4, which in contrast to other pig parvoviruses possesses an additional ORF3, was identified in pigs co-infected with PCV2 in 2010 (Cheung et al., 2010). In 2013 and 2014, PPV5 and PPV6 have been discovered in China and North America (Ni et al., 2014; Schirtzinger et al., 2015; Wu et al., 2014; Xiao et al., 2013c). PPV4, PPV5 and PPV6 are closely related and form a distinct branch based on phylogenetic analysis (Ni et al., 2014; Schirtzinger et al., 2015). However, any possible association with clinical signs of these newly identified PPV genotypes remains unclear.

The objective of this study was to investigate the prevalence rates of PPV1 through PPV6 DNA, PPV1 antibodies and PPV2 antibodies in serum samples collected from 247 pigs located on six farms in Poland during 2013 to 2015.

Materials and Methods

Sample collection

Blood samples (n=247) were collected once (cross-sectional) or in 4-week-intervals (longitudinal) from 2 to 18-week-old pigs from six commercial all-in-all-out pig farms in Poland between 2013 and 2015, were centrifuged, and the serum samples were stored at -20°C. Details on the farms, samples, and sample collections are summarized in Table 1. Farms K, H, PK and P experienced porcine reproductive and respiratory virus (PRRSV) outbreaks before the collection period. Clinical signs suggestive of PPV1 infection were not documented on any of the farms during the collection period.

DNA preparation

Viral DNA was extracted from 50 µl of each serum sample using the MagMax™ Viral RNA isolation kit (Applied Biosystems, Thermo ScientificCarlsbad, CA, USA) on an automated extraction system (Thermo Scientific Kingfisher 96-Deep Well Flex, Waltham, MA, USA) according to the manufacturer’s instructions. The extracted nucleic acids were recovered in 90 µl elution buffer and stored at -20°C until usage.

PCR amplification

Duplex (PPV1 and PPV2) and triplex (PPV3, PPV4 and PPV5) differential real-time PCR assays were performed as described (Opriessnig et al., 2014). In addition, a real-time PCR assay for PPV6 was performed as described (Cui et al., 2016). Amplifications were carried out on a 7500 Fast Real-time PCR system (Applied Biosystems) under the following conditions: initial denaturation at 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Samples with cycle threshold (Ct) values over 37 were considered negative.

Detection of anti-PPV1 and PPV2 antibodies

To further characterize PPV1 and PPV2 in the selected pig populations, antibodies levels were investigated. For PPV1 serum samples were tested by a commercial available blocking ELISA (Ingezim PPV Compac®; Ingenasa, Madrid, Spain) according to the manufacturer’s instructions. For each sample, the blocking index (optical density (OD) negative control – OD sample) / (OD negative control – OD positive control) was calculated. Samples with a blocking index higher than 0.3 were considered positive, samples with a blocking index lower than 0.25 were considered negative and samples with a blocking index between 0.25 and 0.3 were considered suspect.

For PPV2, antibody levels were determined by an in-house ELISA assay. Briefly, purified PPV2 VP2 protein (50 ng per well) was coated on ELISA plates (Nunc® MaxiSorp™, Sigma Aldrich, Roskilde, Denmark) at 4°C overnight. After three washes with phosphate buffered saline with 0.05% Tween 20 (PBST), the plates were blocked with 1% bovine serum albumin for 2 h at 22°C and then incubated with serum diluted 1:100 in PBS containing 10% goat serum at 37°C for 1h. Following three washing steps, the plates were incubated with a 1:10,000 diluted peroxidase-conjugated goat anti-swine IgG (Jackson ImmunoResearch; West Grove, PA, USA) for 1h at 37°C. The peroxidase reaction was visualized by using tetramethylbenzidine-hydrogen peroxide (TMB) (KPL, Gaithersburg, MD, USA) as the substrate for 10 min at room temperature and stopped 50µl of 2N H2SO4. Optical densities (OD) were measured at 405 nm by a Multiskan Ascent 96/384 plate reader (MTX Lab Systems, Vienna, VA, USA). Positive and negative controls were included on each plate. The serum antibody response was presented as sample-to-positive (S/P) ratios calculated as: S/P ratio = (sample OD – negative control mean OD)/(positive control mean OD – negative control mean OD). Samples with an S/P higher or equal to 0.3 were considered positive.

Statistical analysis

Data analysis was performed with SPSS for Windows 22 (SPSS, Chicago, IL, USA). Differences in prevalence were assessed by using the Fisher’s Exact test. A p-value of less than 0.05 was considered statistically significant.

Results

Prevalence rates of the different parvoviruses

The overall parvovirus prevalence rates in piglets and growing pigs on the six farms under investigation were 0% for PPV1, 19% for PPV2, 7.7% for PPV3, 2.4% for PPV4, 4% for PPV5 and 6.1% for PPV6 (Table 2). Parvoviruses were detected more frequently in 9 to 18-week-old pigs compared to 2 to 6-week-old pigs (Table 2, Fig. 1). Of the 173 samples collected from this age group, 79 samples (64.2%) were positive for at least one type of parvovirus. The prevalence rate of PPV2 was the highest with 26.6% (46/173, mean Ct values ± SD 30.4 ± 5.4, range 14.1-36.9), followed by PPV3 at 11% (19/173, 29.9.4 ± 7.4, 14.9-36.8), while the prevalence rates of the other PPV genotypes were below 10% (26.6 ± 6.6). Interestingly, PPV4 was only detected in 14 to 18-week-old pigs and all PPV6 positive samples were detected in 13 to 18-week-old pigs (Fig. 1). Only one 5-week-old pig tested positive for PPV2 and one 6-week-old pig was positive for PPV5 among the 74 serum samples originating from nursery and suckling pigs (Fig. 1).

Co-infection rates for samples with more than one parvovirus

Among the 81 PPV DNA positive samples, one single PPV species was identified in 80.2% (65/81) of the samples with PPV2 being the most frequent identified parvovirus. The remaining 16 PPV DNA positive samples were co-infected with two different genotypes of PPVs (Fig. 1).

Prevalence of PPVs in individual farms

As shown in Fig.1, among the six investigated Polish farms, PPV2 alone was detected in 9 or 10-week-old pigs on Farms H and PK. On Farm R, pigs were positive for PPV2 and PPV5 while in the remaining three farms, P (2015 collection), K and U, all parvoviruses except PPV1 were identified. On Farm K and U, PPV3 through PPV6 were mainly present in grow-finish pigs. The PPV6 prevalence rate in 17 to 18-week-old pigs in these two farms was high (60%, 6/10) when compared to PPV3-5. The peak of PPV2 was observed between 9-13 weeks of age on all farms except Farm K (17 weeks, 20%, 2/10). On Farm P, three PPV genotypes (PPV2, PPV3 and PPV5) were detected in samples collected in 2013/2014. In 2015, PPV4 and PPV6 were also identified.

PPV1 and PPV2 antibody levels

The percentage of PPV2 DNA positive samples and the PPV1 and PPV2 antibody levels on each of the investigated farms are shown in Fig. 2. Seroconversion to PPV1 was commonly at the end nursery or the beginning of the growing stages on most farms. At the first collection at 2 weeks of age all pigs were seronegative for PPV1 (Farm PK, n = 10). PPV1 antibodies were first detected at 5-6 weeks of age (Farm K, H, PK, U, 15/36 positive samples, 22-70% positive samples/farm) or at 9 weeks of age (Farms P-1 and P-2, 4/18, 20-25%). On Farm R all 37 sampled pigs were PPV1 antibodies positive starting with the first collection at 6 weeks of age. In the other farms, the number of PPV1 antibody positive animals increased over time to a prevalence of 100% of positive animals between 12-14 weeks (Farms K, H, PK, P-1, P-2, n = 45) and 18 weeks (Farm U, n = 10). Farm K was the only farm in which the levels of positive animals decreased in the last collection point (8/10 positive animals at 17 weeks of age).

Except for Farm K in which PPV2 DNA was detected in a 5-week-old pig, all 2-to-6-week-old pigs on other investigated farms were negative. The peak of PPV2 DNA detection was between 9-13 weeks of age. PPV2 antibodies were detected in nine of ten 2-week-old pigs. On Farms PK, P-1, P-2, and U, the number of PPV2 antibody positive animals steadily declined from the first collection point at 2-6 weeks of age to 10-13 weeks of age, which is likely consistent with the decline of passively derived immunity. There was a trend for PPV2 seroconversion around 9-13 weeks of age in Farm K, and around 14-17 weeks of age on Farms PK, P-1 and P-2. In Farms H and R, pigs showed moderate levels of PPV2-specific antibodies throughout the collection intervals.

Discussion

Previous studies in Europe have reported variable detection of PPV1-4 (Cadar et al., 2013b; Cságola et al., 2012). In this study, PPV2 through PPV6 were identified, but PPV1 was not detected. In most countries, gilts and sows are routinely and on a regular basis vaccinated to prevent PPV1-affected reproductive failure (Opriessnig et al., 2014). Similarly, all breeding herds who supplied pigs for the investigated farms routinely performed sow PPV1 vaccination. The low PPV1 DNA detection rates may be associated with the vaccination against PPV1 (Ndze et al., 2013) and the short PPV1-viremia period (Opriessnig et al., 2004). In pigs experimentally infected with PPV1, PPV1 DNA was detected in 9/16 pigs at 7 days post inoculation and in 3/16 pigs at 14 days. However, pigs were not positive for PPV1 DNA after 14 days and the mean PPV1-viremia length was only 0.75 (±0.11) weeks (Opriessnig et al., 2004). Similar to Poland, the prevalence of PPV1 in Hungary was also found to be extremely low in growing pigs with only 2 of 392 PPV1 DNA positive samples (0.5%) (Cságola et al., 2012). In another surveillance study in Cameroon, no PPV1 positive sample was identified in 50 stool samples collected in 2011 (Ndze et al., 2013).

In agreement with previous investigations in the USA (Opriessnig et al., 2014), PPV2 was detected most frequently. Among parvovirus positive US samples, 58% (47/81 samples) were PPV2. In the present study, the PPV2 DNA prevalence rate was lower in suckling and nursery pigs (1.4%, 1/74) compared to growing and finisher pigs (26.6%, 46/173). The peak of PPV2 DNA detection was observed in 9 to 13-week-old pigs which is similar to another US surveillance study where PPV2 DNA showed the highest prevalence in grow-finish pigs with low prevalence rates in pigs under 8 weeks of age (Xiao et al., 2013a). The reason for the low detection rates in young pigs may be associated with the protection by PPV2-specific passively-acquired antibodies. All investigated farms showed relatively high levels of PPV2 passively-acquired antibodies at an early age (2-6 weeks) which appeared to decline when the pigs got older. Although little is known about the relevance of PPV2 and clinical signs, reasons for high prevalence of PPV2 in pig herds should be further investigated and any pathogenic potential of PPV2 needs to be determined.

PPV3 has been detected in both wild and domestic pig herds. In wild pigs, high PPV3 prevalence rates of 32.7% and 35% were identified in Germany and Romania, respectively. (Adlhoch et al., 2010; Cadar et al., 2013a). In domestic pigs, the prevalence of PPV3 in the present study was 7.7% (19/247). In contrast, previous studies in other European countries such as Hungary, Romania, Serbia and Croatia and also China high prevalence rates for PPV3 ranging from 9.7% to 47.3% were detected (Cadar et al., 2013b; Cságola et al., 2012; Pan et al., 2012). However, in these studies pig age and farm characteristics were not indicated. Another surveillance carried out in the US demonstrated that PPV3 infection was associated with pig age, no PPV3 DNA was detected in pigs aged under 3 weeks and most PPV3 positive samples were in 8-25 weeks (18.7%, 44/235) (Opriessnig et al., 2014) which was similar to our findings as PPV3 was only detected in 9 to 18-week-old pigs in Poland.

Prevalence of PPV4 in wild pigs was low (only 0.9%) in a previous study (Cadar et al., 2013a). In this study, the overall prevalence of PPV4 in domestic pigs was 2.4%. This is relatively lower than previous reports from Hungary, Romania, Serbia, Croatia, Poland or Cameroon where infection rates ranged from 6.4% to 20%. (Cadar et al., 2013a; Cadar et al., 2013b; Ndze et al., 2013). Maybe due to the overall low numbers of young pigs examined in the current study, all PPV4 positive samples were found in 14 to18-week-old pigs. In contrast, no PPV4 DNA was detected in 2 to 13-week-old pigs.

PPV5 and PPV6 represent very recent in China and the US identified PPV genotypes (Ni et al., 2014; Schirtzinger et al., 2015; Wu et al., 2014; Xiao et al., 2013b), but knowledge of their circulating in Europe is still limited (Cui et al., 2016; Fan et al., 2016). In Poland, the overall prevalence rate of PPV5 was only 4.0%. Most positive samples were identified in grow-finish pigs (9-18 weeks) which is consistent with our previous study in the US (Xiao et al., 2013a). Compared with PPV5, the overall prevalence rates of PPV6 was slightly higher (6.1%). All positive samples for PPV6 were detected in 13 to 18-week-old pigs accounting for a prevalence of 13.6% (15/110) in this age group which is a lower than reported previously (Ni et al., 2014; Schirtzinger et al., 2015). In China 15.6% finishing pigs were positive for PPV6 infection (Ni et al., 2014) and the overall prevalence of PPV6 in North America was similar. In agreement with the findings in Poland from this study, PPV6 was more prevalent than PPV4 and PPV5 in North America (Schirtzinger et al., 2015).

Similar to human PARV4 (Chen et al., 2011), goose parvovirus (Chen et al., 2016) and parvovirus B19 (Puccetti et al., 2012), it is well established that vertical transmission is one of the major transmission routes for PPV1 (Mims, 1981). However, transmission routes of recently identified PPV2 through PPV6 still remain unknown. In agreement with our previous studies (Xiao et al., 2013a; Xiao et al., 2012; Xiao et al., 2013b), most PPV2 through PPV6 DNA positive samples were detected in older pigs (9-18 weeks). Due to the lack of available ELISAs for PPV3, PPV4, PPV5 or PPV6, antibodies levels for these PPVs were not determined in this study. The reason for the low prevalence rates of these PPV types in young pigs below 9 weeks of age may be due to protection by passively-acquired antibodies, a low viremia lengths, or overall low prevalence rates. PPV2 through PPV6 DNA was mainly identified in grow-finish pigs (Fig. 1) and the relatedness of clinical sings and these newly identified parvoviruses should be addressed in future studies. In conclusion, all recently described PPV genotypes were detected on Polish farms and PPV2 was the most frequent PPV genotype circulating in Poland. To our knowledge, this is the first description of identifying PPV2 through PPV6 in pigs in a single European country. Our finding contributes to a better understanding of PPV epidemiology in European pigs.

Funding

Funding was provided by the Biotechnology and Biological Sciences Research Council (BBSRC) Institute Strategic Programme Grant awarded to the Roslin Institute (BB/J004324/1; BBS/E/D/20241864). Dr. Fan was supported by the Program of Study Abroad for Young Teachers by the Agricultural University of Hebei, China. Sample collection in Poland was partially supported by NCN DEC-2013/11/B/NZ7/04950.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure legends

Fig. 1. Prevalence of porcine parvovirus (PPV) types 1, 2, 3, 4, 5 and 6 DNA as determined by real-time PCR assays in serum samples obtained from pigs of different ages housed on one of six commercial pig farms (K, H, U, PK, P and R) located in Poland. Farm P was sampled twice in different years (indicated by the green box). At 11-12 weeks of age, Farm K pigs were moved to a separate fattening facility 300 km away and Farm H pigs were moved 10 km away (indicated by the red boxes). PPV DNA positive samples are indicated by different coloured squares for each PPV type. White squares indicate negative samples. A star in a coloured square indicates a sample with a cycle threshold lower than 25.

Fig. 2. Porcine parvovirus type 2 (PPV2) percentage of real-time PCR positive pigs (left y-axis), PPV2 antibody levels and porcine parvovirus type 1 (PPV1) antibody levels (right y-axis) over time in each of six commercial pig farms (K, H, U, PK, P and R) located in Poland. Farm P was sampled twice in different years: P1 indicates sample collection during 2013/14 and P2 indicates sample collection during 2015.

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