- Email: [email protected]
Prevalence of parvovirus in Minnesota turkeys
Prevalence of parvovirus in Minnesota turkeys T. A. Sharafeldin,∗,†,1 A. Singh,∗ M. Y. Abdel-Glil,∗ S. K. Mor,∗ R. E. Porter,∗ and S. M. Goyal∗ ∗
Department of Veterinary Population Medicine and Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Avenue, St. Paul 55108; and † Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt 44519 LTS flocks, 41 were positive for parvovirus while 20 of 35 samples from non-LTS flocks were positive. The prevalence of parvovirus in fecal samples submitted to MVDL was relatively low; only five of the 116 pools were positive. The partial NS1 gene sequences from LTS and non-LTS samples showed 98 to 100% nt identity except for one divergent turkey parvovirus (TuPV) strain that revealed 90% identity and clustered with chicken-like parvoviruses. The presence of this divergent strain suggests circulation of a recombinant strain of TuPV in Minnesota turkeys. Our results indicate that TuPVs are circulating in both LTS and non-LTS flocks of turkeys in Minnesota, and further experimental studies are indicated to study the role of TuPV in LTS.
Key words: parvovirus, turkeys, poult enteritis, light turkey syndrome, diarrhea, gene sequence 2016 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pew283
INTRODUCTION
these syndromes has been reported during surveillance and experimental studies (Mor et al., 2013). Parvoviruses also have been implicated in turkeys with enteritis and stunting (Trampel et al., 1983) and in chickens with the stunting syndrome (Kisary et al., 1984). In one U.S. study, the prevalence of parvovirus was found to be 77 and 78% in chicken and turkey flocks, respectively (Zsak et al., 2009). In another study, parvovirus prevalence was 71% in commercial turkey flocks in 4 U.S. states (North Carolina, Pennsylvania, Virginia, and Ohio) (Murgia et al., 2012). Initial infection of birds with chicken parvovirus (ChPV) and turkey parvovirus (TuPV) occurs mostly in the first wk of age and the enteric disease occurs between one and 4 wk of age (Day and Zsak, 2013). In goslings and Muscovy ducks, parvovirus causes a fatal disease called the Derzsy’s disease (Kisary and Derzsy, 1974). Recently, parvovirus was implicated in short beak and dwarfism syndrome (SBDS) in mule ducks in France. The family Parvoviridae is divided into 2 subfamilies: Densovirinae, which infects invertebrates, and Parvovirinae, which infects mammals and birds (Tijssen et al., 2011). Parvoviruses are small, icosahedral, nonenveloped particles of ∼20 nm in diameter. The 5 kb genome is linear, single-stranded DNA, which contains 3 open reading frames (5 ORF1, 3 ORF1 and a small ORF located between the major 5 and 3 ORFs).
Poult enteritis complex (PEC) is a general term used for intestinal diseases of young turkeys characterized by enteritis, diarrhea, moderate to marked growth depression, and decreased feed consumption. In certain cases, high mortality may result as is seen in poult enteritis and mortality syndrome (PEMS). Several viruses, bacteria, and protozoa are found to be associated with PEC (Barnes and Guy, 2003). In Minnesota turkeys, poult enteritis syndrome (PES) is a relatively new condition, which affects young turkeys between one d and 7 wk of age. The PES is characterized by diarrhea, depression, and lethargy with pale intestines and excessively fluid cecal contents (Jindal et al., 2009). Another syndrome called the Light Turkey Syndrome (LTS) is a problem of market-age turkeys having lower body weight as compared to their standard breed character (Mor et al., 2013). It is believed that PES at a young age sets up conditions for future development of LTS in older birds (Mor et al., 2013). The occurrence and role of enteric viruses (rotavirus, astrovirus, and reovirus) in C 2016 Poultry Science Association Inc. Received April 7, 2016. Accepted July 1, 2016. 1 Corresponding author: [email protected]
1
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ABSTRACT Poult enteritis syndrome (PES) is characterized by enteritis and decreased body weight gain in growing turkey poults between one d and 7 wk of age. Another syndrome called light turkey syndrome (LTS) causes a decrease in body weight of adult tom turkeys in Minnesota leading to huge economic losses. Reovirus, rotavirus, and astrovirus have been found in LTS and PES flocks in Minnesota. We tested 80 fecal pools collected from four LTS flocks and 35 fecal pools from non-LTS flocks for the presence of parvovirus. In addition, 116 fecal and meconium samples from turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) also were tested. The samples were tested by PCR using primers for the non-structural 1 (NS1) gene of parvovirus. Of the 80 samples from
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The 5 ORF encodes a non-structural protein NS1 while 3 ORF probably encodes the capsid proteins VP1, VP2, and VP3 (Day and Zsak, 2010). Comparative genome sequence analysis of non-structural (NS) gene showed a high degree of homology between ChPV and TuPV (Day and Zsak, 2013). Homology between the classic Derzsy’s disease strain and SBDS strain ranged from 95.9 to 96.8% (Palya et al., 2009). The aim of this study was to compare the prevalence of TuPV in LTS and non-LTS flocks in Minnesota and to determine the presence of this virus in fecal samples submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL). Detection and characterization of TuPV should help in understanding the role of this virus in enteric diseases of Minnesota turkeys.
Samples Fecal samples were collected from 4 commercial turkey flocks in Minnesota. These flocks had historically lower body weights as compared to those of normal flocks and were designated as LTS flocks. Control samples were collected from another 2 flocks with no history of LTS (non-LTS flocks). Fresh fecal samples (approximately one to 2 g each) were collected from 5 different areas (f4 in corners and one in the center) of each barn. The 5 samples from each barn were pooled together in a single pool. Five pooled samples were collected from each flock at 4 different times (2, 3, 5 and 8 wk of age) by the farm staff who was trained for sample collection. These samples were then shipped to the laboratory on ice via a courier. A total of 80 pooled samples from LTS flocks and 35 from non-LTS flocks (pooled samples from one non-LTS flock at 8 wk of age were not collected) were collected during the 4 samplings. The presence of other enteric viruses in these sample pools has been previously described (Mor et al., 2013). Additionally, 116 fecal and meconium samples from poults submitted to the MVDL between 2010 and 2012 also were tested.
DNA Extraction and PCR Individual and pooled fecal samples were homogenized in sterile PBS, pH 7.4 (10% suspension) followed by centrifugation at 2,000 xg for 20 min. The supernatants were then used for extraction of DNA using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). Detection of parvovirus was done using HotStar Taq Master Mix Kit (Qiagen, Valencia, CA) and primers for the non-structural 1 (NS1) gene as described by Zsak et al. (2009). The PCR reaction included 15-min incubation at 95◦ C followed by 30 amplification cycles (94◦ C for 30 s, 55◦ C for one min, and 68◦ C for 3 min). PCR product was visualized on 1.2% agarose gel in Trisacetate-EDTA buffer following electrophoresis. The
DNA Sequencing Of the samples found positive by PCR, 50 (40 from LTS 40 and 10 from non-LTS flocks) were subjected to partial NS1 gene sequencing. Briefly, PCR products were purified using a PCR purification kit (Qiagen, Valencia, CA) and then submitted to the Biomedical Genomic Center, University of Minnesota. By using the same primers as used in PCR reactions, the sequencing was done in both directions. The resulting forward and reverse sequences were aligned together using Sequencher software (www.msi.umn.edu). The BLAST analysis was done using the NCBI BLAST (www.ncbi.nlm.nih.gov). The obtained nucleotide sequences were aligned by the Clustal W method using MEGA 6.0 software. Maximum Likelihood method based on general time-reversible (GTR) model with 1000 bootstraps was used for computing the phylogenetic tree of aligned sequences, and evolutionary distances was constructed by P-distance method.
Nucleotide Sequence Accession Numbers The obtained partial sequences (n = 50) were submitted to the GenBank database under the following accession numbers: KU569242-KU569291.
RESULTS Of the 80 fecal pools from 4 LTS flocks, 51% (41/80) were positive for TuPV by PCR. The percent of positive samples was higher at 2 and 5 wk (85 to 90%) of age. At 8 wk of age, 3 of the 4 LTS flocks were PCR negative except for one flock that had 80% (4/5) of samples positive. Of the 35 fecal pools collected from the 2 nonLTS flock, 57% (20/35) were PCR positive. There was no virus positive sample at first sampling in either of the 2 non-LTS flocks while the rate of positive samples increased gradually to 100% at 5 and 8 wk of age. Of the 116 diagnostic samples of feces and meconium, only 5 fecal samples were positive by PCR (Table 1). The partial gene sequence and phylogenetic analysis of NS1 gene showed 90 to 100% similarity among TuPVMN strains from LTS flocks (n = 40) and a reference U.S. strain (TuPV-1078). Strains from non-LTS flocks (n = 10) showed 99 to 100% nt similarity with that of the reference strain. On phylogenetic analysis, all but one strain (TuPV-MN30) clustered in the TuPV-like group along with TuPV strains from the United States and Hungary. Surprisingly, TuPV-MN30 was clustered in a separate lineage (>10% divergence) along with chicken parvovirus-like strains from the United States. TuPV-MN showed no homogeneity with duck and goose parvoviruses (Figure 1).
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MATERIALS AND METHODS
position of the band on agarose gel at 560 bp confirmed the presence of parvovirus.
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PARVOVIRUS IN TURKEYS Table 1. Fecal pools positive for turkey parvovirus by PCR. Number (%) positive ina LTS flocks Age 2 3 5 8
wk wk wk wk
Non-LTS flocks
Flock 1
Flock 2
Flock 3
Flock 4
Flock 5
Flock 6
5/5 (100%) 2/5 (40%) 4/5 (80%) 0/5 (0%)
5/5 (100%) 0/5 (0%) 4/5 (80%) 0/5 (0%)
3/5 (60%) 0/5 (0%) 5/5 (100%) 0/5 (0%)
4/5 (80%) 0/5 (0%) 5/5 (100%) 4/5 (80%)
0/5 (0%) 3/5 (60%) 5/5 (100%) 5/5 (100%)
0/5 (0%) 2/5 (40%) 5/5 (100%) NS
a LTS = Light Turkey Syndrome; Non-LTS = Non-Light Turkey Syndrome. NS: Not tested.
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Figure 1. Phylogenetic tree of 50 Minnesota TuPV strains based on partial nucleotide sequences (560 bp) of NS1 gene. Phylogenetic tree was constructed using the Maximum Likelihood statistical method based on general time-reversible (GTR) model with 1000 bootstraps (sequences of study are in bold).
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DISCUSSION
REFERENCES Barnes, H. J., and J. S. Guy. 2003. Poult enteritis mortality syndrome. Pages 1171–1180 in Diseases of Poultry, 11th ed. Y. M. Saif, H. J. Barnes, A. Fadly, J. R. Glisson, L. R. McDougald, and D. E. Swayne, Eds. Iowa state press. Day, J. M., and L. Zsak. 2010. Determination and analysis of the full length chicken parvovirus genome. Virol. J. 399:59–64.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Australia on October 6, 2016
The presence of RNA viruses such as astrovirus, rotavirus, and reovirus in association with LTS has been reported previously (Mor et al., 2013). However, an association of DNA viruses with LTS and PES has not been investigated although parvovirus infections have been described in broiler flocks displaying runtingstunting syndrome (RSS) and in young turkeys with PEMS in Hungary (Palade et al., 2011). The virus has been detected in intestinal contents as well as epithelial and inflammatory cells of lamina propria of the duodenum and jejunum of both chickens and turkeys. The virus also has been detected in the bursa of Fabricius, liver, and exocrine pancreas of turkeys (Palade et al., 2011). Serious growth retardation, bad feathering, and bone disorders were seen after experimental infection of commercial and SPF broilers and turkey poults with ChPV ABU-P1 strain and TuPV 1078, respectively (Zsak and Day, 2010a,b). Most of the PCR positive samples in LTS birds were at 2 and 5 wk of age. In non-LTS flocks, the percentage of positive samples increased gradually up to 100% at 8 wk of age. This pattern of viral detection may refer to a possible role of early parvovirus infection in PES and LTS in Minnesota. Domanska-Blicharz et al. (2012) found 58 of 194 turkey flocks (29.4%) positive for parvovirus in Poland. Most of the positive flocks were suffering from PEC while 7 flocks had PEMS and another 3 flocks did not show any clinical enteritis. The age of positive flocks was one to 19 wk with a majority being positive at 3 to 7 wk. The higher prevalence of parvovirus in Minnesota turkeys is probably because the samples were taken from young poults of 2 to 8 wk of age. In a previous study, Zsak et al. (2009) found the prevalence of TuPV in the United States to be 78% in poults up to 7 wk of age. Murgia et al. (2012) found an overall parvovirus prevalence of 71.6% in fecal samples of turkeys collected from 4 different U.S. states (Virginia, North Carolina, Pennsylvania, and Ohio). In general, they observed 20% lower prevalence in poults less than 7 wk of age as compared to the results reported by Zsak et al. (2009). Our results are similar to those obtained by Murgia et al. (2012) for less than 8-week-old poults. Of the samples submitted to the MVDL for disease diagnosis, meconium samples were from one d of age and fecal samples were from older ages (one to 5 wk). The 5 positive samples were only in fecal samples while all meconium samples were negative. This suggests that the virus infects birds after hatch and the way of transmission is probably horizontal rather than vertical. Further experimental studies are needed to verify this suggestion. The partial NS1 gene sequences of the selected LTS and non-LTS turkey fecal samples were compared with those of 4 ChPV (including Canadian ChPV), 5 TuPV (including Polish and Hungarian strains), and 6 goose and duck parvoviruses retrieved from GenBank. The
homogeneity of TuPV-MN strains with one TuPV in the United States, a parvovirus from Poland, and a ChPV from Canada ranged from 98 to 100%. The similarity with turkey parvovirus from Hungary was 96.6 to 97.7%. These results suggest a close relationship between TuPV-MN strains and the Canadian ChPV. These results are also in close agreement with a previous report in which the percent nucleotide identity among U.S. turkey strains was between 98.4 and 100% (Murgia et al., 2012). The TuPV-MN30 strain clustered with some TuPV and ChPV strains from the United States in a different lineage with >10 divergence. Another study also reported similar findings in which the NS1 gene of 5 TuPV and 5 ChPV clustered separately except for one TuPV and one ChPV strain, which clustered in opposite groups. Interestingly, these 2 divergent strains were also from Minnesota turkeys (Zsak et al., 2015). These data indicate that recombinant-like strains of TuPV are circulating in Minnesota turkeys. Further studies are recommended to determine their true prevalence. Day and Zsak (2010) analyzed the full-length genomes of one ChPV and 2 TuPV and showed the presence of high sequence similarity between chicken and turkey parvoviruses. However, our study and others (Zsak et al., 2009; Palade et al., 2011) based on the analysis of partial sequence of NS gene, showed that the chicken and turkey viruses cluster separately, which suggests that they are probably divergent viruses from a common ancestor. The similarity between TuPV and some ChPV, as observed in our study, may be attributed to interspecies transmission of parvoviruses (Murgia et al., 2012). Future experimental transmission studies are needed to confirm this hypothesis. In conclusion, TuPV prevalence in Minnesota turkey flocks with LTS and non-LTS was 51 and 57%, respectively. The virus was detected mostly at an early age in LTS flocks and at a later age in non-LTS flocks, suggesting a possible role of early parvovirus infection in PES and LTS. The virus infects birds after hatching and vertical transmission appears unlikely. Further experimental studies are needed to verify these hypotheses. In addition, the homogeneity of detected TuPV-MN strains with another TuPV in the United States and Europe and absence of homogeneity with goose and duck parvovirus confirms the relationship of TuPV-MN strains to TuPV lineages. The presence of a separate lineage cluster of TuPV and ChPV may be suggestive of possible mutations. Complete genome sequencing in the future may approve or reject this suggestion.
PARVOVIRUS IN TURKEYS
Palya, V., A. Zolnai, Z. Benyeda, E. Kov´ acs, V. Kardi, and T. Mat´ o. 2009. Short beak and dwarfism syndrome of mule duck is caused by a distinct lineage of goose parvovirus. Avian Pathol. 38:175– 180. Tijssen, P., M. Agbandje-McKenna, J. M. Almendral, Bergoin, T. W. Flegel, K. Hedman, J. Kleinschmidt, Y. Li, D. J. Pintel, and P. Tattersall. 2011. The family Parvoviridae. Pages 405–425 in Virus taxonomy—Ninth Report of the International Committee on Taxonomy of Viruses. A. M. Q. King, M. J. Adams, E. B. Carstens, and E. J. Lefkowitz, Eds. Elsevier/Academic Press, London. Trampel, D. W., D. A. Kinden, R. F. Solorzano, and P. L. Stogsdill. 1983. Parvovirus-like enteropathy in Missouri turkeys. Avian Dis. 27:49–54. Zsak, L., and J. M. Day. 2010a. Parvovirus-induced Enteritis in Young Turkeys. Proc. 59th West. Poult. Dis. Conf., Vancouver, Canada. Page 22. Zsak, L., and J. M. Day. 2010b. The Pathogenicity of Parvovirus in Chickens. Proc. Amer. Ass. Avian Pathol. Conf., Atlanta, GA. Pages 92–93. Zsak, L., K. O. Strother, and J. M. Day. 2009. Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses. Avian Dis. 53:83–88. Zsak, L., A. R. M. Cha, F. Li, and J. M. Day. 2015. Host specificity and phylogenetic relationships of chicken and turkey parvoviruses. Avian Dis. 59:157–161.
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Day, J. M., and L. Zsak. 2013. Recent progress in the characterization of avian enteric viruses. Avian Dis. 57:573–580. Domanska-Blicharz, K., A. Jacukowicz, A. Lisowska, and Z. Minta. 2012. Genetic characterization of parvoviruses circulating in turkey and chicken flocks in Poland. Arch. Virol. 157:2425– 2430. Jindal, N., D. P. Patnayak, A. Ziegler, A. Lago, and S. M. Goyal. 2009. A retrospective study on poult enteritis syndrome in Minnesota. Avian Dis. 53:268–275. Kisary, J., and D. Derzsy. 1974. Viral disease of goslings. IV. Characterization of the causal agent in tissue culture system. Acta. Vet. Acad. Sci. Hung. 24:287–292. Kisary, J., B. Nagy, and Z. Bitay. 1984. Presence of parvoviruses in the intestine of chickens showing stunting syndrome. Avian Pathol. 13:339–343. Mor, S. K., T. A. Sharafeldin, M. Abin, M. Kromm, R. E. Porter, S. M. Goyal, and D. P. Patnayak. 2013. The occurrence of enteric viruses in light turkey syndrome. Avian Pathol. 42:497–501. Murgia, M., A. Rauf, Y. Tang, E. Gingerich, C. W. Lee, and Y. M. Saif. 2012. Prevalence of parvoviruses in commercial turkey flocks. Avian Dis. 56:744–749. Palade, E. A., Z. Demeter, A. Hornyak, C. Nemes, J. Kisary, and M. Rusvai. 2011. High prevalence of turkey parvovirus in turkey flocks from Hungary experiencing enteric disease syndromes. Avian Dis. 55:468–475.
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Department of Veterinary Population Medicine and Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, 1333 Gortner Avenue, St. Paul 55108; and † Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt 44519 LTS flocks, 41 were positive for parvovirus while 20 of 35 samples from non-LTS flocks were positive. The prevalence of parvovirus in fecal samples submitted to MVDL was relatively low; only five of the 116 pools were positive. The partial NS1 gene sequences from LTS and non-LTS samples showed 98 to 100% nt identity except for one divergent turkey parvovirus (TuPV) strain that revealed 90% identity and clustered with chicken-like parvoviruses. The presence of this divergent strain suggests circulation of a recombinant strain of TuPV in Minnesota turkeys. Our results indicate that TuPVs are circulating in both LTS and non-LTS flocks of turkeys in Minnesota, and further experimental studies are indicated to study the role of TuPV in LTS.
Key words: parvovirus, turkeys, poult enteritis, light turkey syndrome, diarrhea, gene sequence 2016 Poultry Science 0:1–5 http://dx.doi.org/10.3382/ps/pew283
INTRODUCTION
these syndromes has been reported during surveillance and experimental studies (Mor et al., 2013). Parvoviruses also have been implicated in turkeys with enteritis and stunting (Trampel et al., 1983) and in chickens with the stunting syndrome (Kisary et al., 1984). In one U.S. study, the prevalence of parvovirus was found to be 77 and 78% in chicken and turkey flocks, respectively (Zsak et al., 2009). In another study, parvovirus prevalence was 71% in commercial turkey flocks in 4 U.S. states (North Carolina, Pennsylvania, Virginia, and Ohio) (Murgia et al., 2012). Initial infection of birds with chicken parvovirus (ChPV) and turkey parvovirus (TuPV) occurs mostly in the first wk of age and the enteric disease occurs between one and 4 wk of age (Day and Zsak, 2013). In goslings and Muscovy ducks, parvovirus causes a fatal disease called the Derzsy’s disease (Kisary and Derzsy, 1974). Recently, parvovirus was implicated in short beak and dwarfism syndrome (SBDS) in mule ducks in France. The family Parvoviridae is divided into 2 subfamilies: Densovirinae, which infects invertebrates, and Parvovirinae, which infects mammals and birds (Tijssen et al., 2011). Parvoviruses are small, icosahedral, nonenveloped particles of ∼20 nm in diameter. The 5 kb genome is linear, single-stranded DNA, which contains 3 open reading frames (5 ORF1, 3 ORF1 and a small ORF located between the major 5 and 3 ORFs).
Poult enteritis complex (PEC) is a general term used for intestinal diseases of young turkeys characterized by enteritis, diarrhea, moderate to marked growth depression, and decreased feed consumption. In certain cases, high mortality may result as is seen in poult enteritis and mortality syndrome (PEMS). Several viruses, bacteria, and protozoa are found to be associated with PEC (Barnes and Guy, 2003). In Minnesota turkeys, poult enteritis syndrome (PES) is a relatively new condition, which affects young turkeys between one d and 7 wk of age. The PES is characterized by diarrhea, depression, and lethargy with pale intestines and excessively fluid cecal contents (Jindal et al., 2009). Another syndrome called the Light Turkey Syndrome (LTS) is a problem of market-age turkeys having lower body weight as compared to their standard breed character (Mor et al., 2013). It is believed that PES at a young age sets up conditions for future development of LTS in older birds (Mor et al., 2013). The occurrence and role of enteric viruses (rotavirus, astrovirus, and reovirus) in C 2016 Poultry Science Association Inc. Received April 7, 2016. Accepted July 1, 2016. 1 Corresponding author: [email protected]
1
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ABSTRACT Poult enteritis syndrome (PES) is characterized by enteritis and decreased body weight gain in growing turkey poults between one d and 7 wk of age. Another syndrome called light turkey syndrome (LTS) causes a decrease in body weight of adult tom turkeys in Minnesota leading to huge economic losses. Reovirus, rotavirus, and astrovirus have been found in LTS and PES flocks in Minnesota. We tested 80 fecal pools collected from four LTS flocks and 35 fecal pools from non-LTS flocks for the presence of parvovirus. In addition, 116 fecal and meconium samples from turkeys submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) also were tested. The samples were tested by PCR using primers for the non-structural 1 (NS1) gene of parvovirus. Of the 80 samples from
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SHARAFELDIN ET AL.
The 5 ORF encodes a non-structural protein NS1 while 3 ORF probably encodes the capsid proteins VP1, VP2, and VP3 (Day and Zsak, 2010). Comparative genome sequence analysis of non-structural (NS) gene showed a high degree of homology between ChPV and TuPV (Day and Zsak, 2013). Homology between the classic Derzsy’s disease strain and SBDS strain ranged from 95.9 to 96.8% (Palya et al., 2009). The aim of this study was to compare the prevalence of TuPV in LTS and non-LTS flocks in Minnesota and to determine the presence of this virus in fecal samples submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL). Detection and characterization of TuPV should help in understanding the role of this virus in enteric diseases of Minnesota turkeys.
Samples Fecal samples were collected from 4 commercial turkey flocks in Minnesota. These flocks had historically lower body weights as compared to those of normal flocks and were designated as LTS flocks. Control samples were collected from another 2 flocks with no history of LTS (non-LTS flocks). Fresh fecal samples (approximately one to 2 g each) were collected from 5 different areas (f4 in corners and one in the center) of each barn. The 5 samples from each barn were pooled together in a single pool. Five pooled samples were collected from each flock at 4 different times (2, 3, 5 and 8 wk of age) by the farm staff who was trained for sample collection. These samples were then shipped to the laboratory on ice via a courier. A total of 80 pooled samples from LTS flocks and 35 from non-LTS flocks (pooled samples from one non-LTS flock at 8 wk of age were not collected) were collected during the 4 samplings. The presence of other enteric viruses in these sample pools has been previously described (Mor et al., 2013). Additionally, 116 fecal and meconium samples from poults submitted to the MVDL between 2010 and 2012 also were tested.
DNA Extraction and PCR Individual and pooled fecal samples were homogenized in sterile PBS, pH 7.4 (10% suspension) followed by centrifugation at 2,000 xg for 20 min. The supernatants were then used for extraction of DNA using the QIAamp DNA Stool Mini Kit (Qiagen, Valencia, CA). Detection of parvovirus was done using HotStar Taq Master Mix Kit (Qiagen, Valencia, CA) and primers for the non-structural 1 (NS1) gene as described by Zsak et al. (2009). The PCR reaction included 15-min incubation at 95◦ C followed by 30 amplification cycles (94◦ C for 30 s, 55◦ C for one min, and 68◦ C for 3 min). PCR product was visualized on 1.2% agarose gel in Trisacetate-EDTA buffer following electrophoresis. The
DNA Sequencing Of the samples found positive by PCR, 50 (40 from LTS 40 and 10 from non-LTS flocks) were subjected to partial NS1 gene sequencing. Briefly, PCR products were purified using a PCR purification kit (Qiagen, Valencia, CA) and then submitted to the Biomedical Genomic Center, University of Minnesota. By using the same primers as used in PCR reactions, the sequencing was done in both directions. The resulting forward and reverse sequences were aligned together using Sequencher software (www.msi.umn.edu). The BLAST analysis was done using the NCBI BLAST (www.ncbi.nlm.nih.gov). The obtained nucleotide sequences were aligned by the Clustal W method using MEGA 6.0 software. Maximum Likelihood method based on general time-reversible (GTR) model with 1000 bootstraps was used for computing the phylogenetic tree of aligned sequences, and evolutionary distances was constructed by P-distance method.
Nucleotide Sequence Accession Numbers The obtained partial sequences (n = 50) were submitted to the GenBank database under the following accession numbers: KU569242-KU569291.
RESULTS Of the 80 fecal pools from 4 LTS flocks, 51% (41/80) were positive for TuPV by PCR. The percent of positive samples was higher at 2 and 5 wk (85 to 90%) of age. At 8 wk of age, 3 of the 4 LTS flocks were PCR negative except for one flock that had 80% (4/5) of samples positive. Of the 35 fecal pools collected from the 2 nonLTS flock, 57% (20/35) were PCR positive. There was no virus positive sample at first sampling in either of the 2 non-LTS flocks while the rate of positive samples increased gradually to 100% at 5 and 8 wk of age. Of the 116 diagnostic samples of feces and meconium, only 5 fecal samples were positive by PCR (Table 1). The partial gene sequence and phylogenetic analysis of NS1 gene showed 90 to 100% similarity among TuPVMN strains from LTS flocks (n = 40) and a reference U.S. strain (TuPV-1078). Strains from non-LTS flocks (n = 10) showed 99 to 100% nt similarity with that of the reference strain. On phylogenetic analysis, all but one strain (TuPV-MN30) clustered in the TuPV-like group along with TuPV strains from the United States and Hungary. Surprisingly, TuPV-MN30 was clustered in a separate lineage (>10% divergence) along with chicken parvovirus-like strains from the United States. TuPV-MN showed no homogeneity with duck and goose parvoviruses (Figure 1).
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MATERIALS AND METHODS
position of the band on agarose gel at 560 bp confirmed the presence of parvovirus.
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PARVOVIRUS IN TURKEYS Table 1. Fecal pools positive for turkey parvovirus by PCR. Number (%) positive ina LTS flocks Age 2 3 5 8
wk wk wk wk
Non-LTS flocks
Flock 1
Flock 2
Flock 3
Flock 4
Flock 5
Flock 6
5/5 (100%) 2/5 (40%) 4/5 (80%) 0/5 (0%)
5/5 (100%) 0/5 (0%) 4/5 (80%) 0/5 (0%)
3/5 (60%) 0/5 (0%) 5/5 (100%) 0/5 (0%)
4/5 (80%) 0/5 (0%) 5/5 (100%) 4/5 (80%)
0/5 (0%) 3/5 (60%) 5/5 (100%) 5/5 (100%)
0/5 (0%) 2/5 (40%) 5/5 (100%) NS
a LTS = Light Turkey Syndrome; Non-LTS = Non-Light Turkey Syndrome. NS: Not tested.
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Figure 1. Phylogenetic tree of 50 Minnesota TuPV strains based on partial nucleotide sequences (560 bp) of NS1 gene. Phylogenetic tree was constructed using the Maximum Likelihood statistical method based on general time-reversible (GTR) model with 1000 bootstraps (sequences of study are in bold).
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DISCUSSION
REFERENCES Barnes, H. J., and J. S. Guy. 2003. Poult enteritis mortality syndrome. Pages 1171–1180 in Diseases of Poultry, 11th ed. Y. M. Saif, H. J. Barnes, A. Fadly, J. R. Glisson, L. R. McDougald, and D. E. Swayne, Eds. Iowa state press. Day, J. M., and L. Zsak. 2010. Determination and analysis of the full length chicken parvovirus genome. Virol. J. 399:59–64.
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The presence of RNA viruses such as astrovirus, rotavirus, and reovirus in association with LTS has been reported previously (Mor et al., 2013). However, an association of DNA viruses with LTS and PES has not been investigated although parvovirus infections have been described in broiler flocks displaying runtingstunting syndrome (RSS) and in young turkeys with PEMS in Hungary (Palade et al., 2011). The virus has been detected in intestinal contents as well as epithelial and inflammatory cells of lamina propria of the duodenum and jejunum of both chickens and turkeys. The virus also has been detected in the bursa of Fabricius, liver, and exocrine pancreas of turkeys (Palade et al., 2011). Serious growth retardation, bad feathering, and bone disorders were seen after experimental infection of commercial and SPF broilers and turkey poults with ChPV ABU-P1 strain and TuPV 1078, respectively (Zsak and Day, 2010a,b). Most of the PCR positive samples in LTS birds were at 2 and 5 wk of age. In non-LTS flocks, the percentage of positive samples increased gradually up to 100% at 8 wk of age. This pattern of viral detection may refer to a possible role of early parvovirus infection in PES and LTS in Minnesota. Domanska-Blicharz et al. (2012) found 58 of 194 turkey flocks (29.4%) positive for parvovirus in Poland. Most of the positive flocks were suffering from PEC while 7 flocks had PEMS and another 3 flocks did not show any clinical enteritis. The age of positive flocks was one to 19 wk with a majority being positive at 3 to 7 wk. The higher prevalence of parvovirus in Minnesota turkeys is probably because the samples were taken from young poults of 2 to 8 wk of age. In a previous study, Zsak et al. (2009) found the prevalence of TuPV in the United States to be 78% in poults up to 7 wk of age. Murgia et al. (2012) found an overall parvovirus prevalence of 71.6% in fecal samples of turkeys collected from 4 different U.S. states (Virginia, North Carolina, Pennsylvania, and Ohio). In general, they observed 20% lower prevalence in poults less than 7 wk of age as compared to the results reported by Zsak et al. (2009). Our results are similar to those obtained by Murgia et al. (2012) for less than 8-week-old poults. Of the samples submitted to the MVDL for disease diagnosis, meconium samples were from one d of age and fecal samples were from older ages (one to 5 wk). The 5 positive samples were only in fecal samples while all meconium samples were negative. This suggests that the virus infects birds after hatch and the way of transmission is probably horizontal rather than vertical. Further experimental studies are needed to verify this suggestion. The partial NS1 gene sequences of the selected LTS and non-LTS turkey fecal samples were compared with those of 4 ChPV (including Canadian ChPV), 5 TuPV (including Polish and Hungarian strains), and 6 goose and duck parvoviruses retrieved from GenBank. The
homogeneity of TuPV-MN strains with one TuPV in the United States, a parvovirus from Poland, and a ChPV from Canada ranged from 98 to 100%. The similarity with turkey parvovirus from Hungary was 96.6 to 97.7%. These results suggest a close relationship between TuPV-MN strains and the Canadian ChPV. These results are also in close agreement with a previous report in which the percent nucleotide identity among U.S. turkey strains was between 98.4 and 100% (Murgia et al., 2012). The TuPV-MN30 strain clustered with some TuPV and ChPV strains from the United States in a different lineage with >10 divergence. Another study also reported similar findings in which the NS1 gene of 5 TuPV and 5 ChPV clustered separately except for one TuPV and one ChPV strain, which clustered in opposite groups. Interestingly, these 2 divergent strains were also from Minnesota turkeys (Zsak et al., 2015). These data indicate that recombinant-like strains of TuPV are circulating in Minnesota turkeys. Further studies are recommended to determine their true prevalence. Day and Zsak (2010) analyzed the full-length genomes of one ChPV and 2 TuPV and showed the presence of high sequence similarity between chicken and turkey parvoviruses. However, our study and others (Zsak et al., 2009; Palade et al., 2011) based on the analysis of partial sequence of NS gene, showed that the chicken and turkey viruses cluster separately, which suggests that they are probably divergent viruses from a common ancestor. The similarity between TuPV and some ChPV, as observed in our study, may be attributed to interspecies transmission of parvoviruses (Murgia et al., 2012). Future experimental transmission studies are needed to confirm this hypothesis. In conclusion, TuPV prevalence in Minnesota turkey flocks with LTS and non-LTS was 51 and 57%, respectively. The virus was detected mostly at an early age in LTS flocks and at a later age in non-LTS flocks, suggesting a possible role of early parvovirus infection in PES and LTS. The virus infects birds after hatching and vertical transmission appears unlikely. Further experimental studies are needed to verify these hypotheses. In addition, the homogeneity of detected TuPV-MN strains with another TuPV in the United States and Europe and absence of homogeneity with goose and duck parvovirus confirms the relationship of TuPV-MN strains to TuPV lineages. The presence of a separate lineage cluster of TuPV and ChPV may be suggestive of possible mutations. Complete genome sequencing in the future may approve or reject this suggestion.
PARVOVIRUS IN TURKEYS
Palya, V., A. Zolnai, Z. Benyeda, E. Kov´ acs, V. Kardi, and T. Mat´ o. 2009. Short beak and dwarfism syndrome of mule duck is caused by a distinct lineage of goose parvovirus. Avian Pathol. 38:175– 180. Tijssen, P., M. Agbandje-McKenna, J. M. Almendral, Bergoin, T. W. Flegel, K. Hedman, J. Kleinschmidt, Y. Li, D. J. Pintel, and P. Tattersall. 2011. The family Parvoviridae. Pages 405–425 in Virus taxonomy—Ninth Report of the International Committee on Taxonomy of Viruses. A. M. Q. King, M. J. Adams, E. B. Carstens, and E. J. Lefkowitz, Eds. Elsevier/Academic Press, London. Trampel, D. W., D. A. Kinden, R. F. Solorzano, and P. L. Stogsdill. 1983. Parvovirus-like enteropathy in Missouri turkeys. Avian Dis. 27:49–54. Zsak, L., and J. M. Day. 2010a. Parvovirus-induced Enteritis in Young Turkeys. Proc. 59th West. Poult. Dis. Conf., Vancouver, Canada. Page 22. Zsak, L., and J. M. Day. 2010b. The Pathogenicity of Parvovirus in Chickens. Proc. Amer. Ass. Avian Pathol. Conf., Atlanta, GA. Pages 92–93. Zsak, L., K. O. Strother, and J. M. Day. 2009. Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses. Avian Dis. 53:83–88. Zsak, L., A. R. M. Cha, F. Li, and J. M. Day. 2015. Host specificity and phylogenetic relationships of chicken and turkey parvoviruses. Avian Dis. 59:157–161.
Downloaded from http://ps.oxfordjournals.org/ at University of Western Australia on October 6, 2016
Day, J. M., and L. Zsak. 2013. Recent progress in the characterization of avian enteric viruses. Avian Dis. 57:573–580. Domanska-Blicharz, K., A. Jacukowicz, A. Lisowska, and Z. Minta. 2012. Genetic characterization of parvoviruses circulating in turkey and chicken flocks in Poland. Arch. Virol. 157:2425– 2430. Jindal, N., D. P. Patnayak, A. Ziegler, A. Lago, and S. M. Goyal. 2009. A retrospective study on poult enteritis syndrome in Minnesota. Avian Dis. 53:268–275. Kisary, J., and D. Derzsy. 1974. Viral disease of goslings. IV. Characterization of the causal agent in tissue culture system. Acta. Vet. Acad. Sci. Hung. 24:287–292. Kisary, J., B. Nagy, and Z. Bitay. 1984. Presence of parvoviruses in the intestine of chickens showing stunting syndrome. Avian Pathol. 13:339–343. Mor, S. K., T. A. Sharafeldin, M. Abin, M. Kromm, R. E. Porter, S. M. Goyal, and D. P. Patnayak. 2013. The occurrence of enteric viruses in light turkey syndrome. Avian Pathol. 42:497–501. Murgia, M., A. Rauf, Y. Tang, E. Gingerich, C. W. Lee, and Y. M. Saif. 2012. Prevalence of parvoviruses in commercial turkey flocks. Avian Dis. 56:744–749. Palade, E. A., Z. Demeter, A. Hornyak, C. Nemes, J. Kisary, and M. Rusvai. 2011. High prevalence of turkey parvovirus in turkey flocks from Hungary experiencing enteric disease syndromes. Avian Dis. 55:468–475.
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