STUDIES ON TURKEY PARVOVIRUSES

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Maria Vittoria Murgia

Graduate Program in Veterinary Preventive Medicine

The Ohio State University

2012

Dissertation Committee:

Dr. Y. M. Saif (Advisor) Dr. D. J. Jackwood Dr. C-W Lee Dr. J. LeJeune

Copyrighted by

Maria Vittoria Murgia

2012

Abstract

Turkey parvovirus belongs to the family Parvoviridae, subfamily Parvovirinae, genus parvovirus. It was identified in turkeys with enteritis for the first time in 1983. Since then there were no further reports on turkey parvovirus in the United States (US) until 2008 when Zsak et al. determined the partial sequence of the non structural (NS) gene of chicken and turkey parvoviruses and then in 2009 developed a PCR which was used to test fecal samples collected from various US states. A high prevalence of parvoviruses was detected by PCR in that study; however there was no information on the health status of the flocks from which the samples were collected. Moreover, there is no information regarding the pathogenesis of turkey parvoviruses and their involvement in the enteric diseases of poultry. The objectives of our studies were:

1. To determine the prevalence of parvovirus in various US states in different years

2. To determine the presence of other viruses in turkey poults in conjunction with

parvoviruses

3. To develop a sensitive diagnostic assay for the detection of turkey parvoviruses in

fecal samples

4. To determine the pathogenicity of parvovirus in SPF turkey poults.

Intestinal content, feces or litter samples collected between 2000 and 2010 in four different states (Virginia, North Carolina, Pennsylvania, and Ohio) were used to address

ii the first two objectives. Those samples were tested for parvovirus and other enteric viruses using PCR and RT-PCR with previously published primers and also with transmission electron microscopy. The overall prevalence of parvovirus (71.5%) was comparable with the previous studies. In our study, we had samples collected from birds of a wider age range and we compared the prevalence of parvoviruses in two age groups,

1-7 weeks and 8-19 weeks of age. We found that although parvovirus was widespread in both age groups, a significantly higher prevalence was detected in the older birds.

Moreover, in the majority of the cases, parvovirus was detected in conjunction with other enteric viruses, such as astrovirus, reovirus and rotavirus. To support future surveillance and research, we develop Real-Time PCR test targeting a conserved region of the NS gene. This test showed the same analytical sensitivity and specificity compared to conventional PCR test and good intra and inter-assay repeatability. Moreover, it was shown to detect parvovirus’ DNA in field fecal samples. The real-time PCR is faster and it is possible to quantify the DNA which are advantages compared to the conventional

PCR. Since we were not able to purify parvovirus, we determine the pathogenesis of parvovirus in combination with astrovirus and compare the outcome to that of astrovirus alone. In two different in vivo trials using 2-4 week-old SPF turkeys, we observed early replication of astrovirus in intestine and the parvovirus was detected in later time points when astrovirus shedding decreased. Our study shows potential viral interference among different enteric viruses and also shed light into the persistence or higher prevalence of parvovirus in turkeys of older ages in the field compared to astrovirus.

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Dedicated to my parents, my brother and my sister

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Acknowledgments

I am very obliged to my advisor Dr. Yehia M. Saif for his guidance and support throughout my studies. He showed me how to look at everything with a positive attitude. He had always words of encouragement especially when the experiments did not work as expected. While working in his lab under his supervision I have grown as a scientist as well as a person and I will treasure this great experience for all of my life. I am grateful to my committee members Dr. Daral J. Jackwood, Dr. Chang-Won Lee, and Dr. Jeff LeJeune for their suggestions and comments.

I am very thankful to Dr. Qiuhong Wang for her precious suggestions and comments. She always kept her door open when I needed her opinion.

I would like to thank my labmates Dr. Abdul Rauf and former labmates Dr. Hadi Yassine and Dr. Yuxin Tang for their technical support and friendship. I am very thankful to Dr. Alex Rodriguez-Palaci and Dr. Kwonil Jung for their technical support and suggestions. I would like to thank Dr. Juliette Hanson, Kingsly Belin, Andrew Wright, Greg Myers, and Todd Root for their help with the animal care. I am very thankful to Ms. Robin Weimer and Ms. Hannah Gehman for their kindness and help during all these years.

I am thankful to my fiancé Christian Cruz and his family for sharing with me this experience

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Last but not least, I am obliged to my parents, Ms. Anna Franca Diana and Mr. Severino Murgia, by brother, Francesco, and my sister, Valeria, for supporting always my choices and for being close to me even if there is an ocean that physically separates us. I am appreciative to my aunt Mrs. Silvana Murgia, my grandmas Ms. Maria Mameli and Ms. Bonaria Argiolas for their encouragements throughout my life. I also would like to remember my grandpa Mr. Salvatore Murgia, which passed away few years ago, for his smile and his words of support.

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Vita

July 1996 ...... High School Diploma,

...... Liceo Classico “De Castro”, Oristano, Italy

October 2003 ...... Laurea in Biotechnology, Bologna

...... University, Bologna, Italy

October to March 2004 ...... Graduate visitor at the Virology Laboratory,

...... Department of Veterinary Public Health and

...... Animal Pathology, University of Bologna,

...... Bologna, Italy

March 2003 to July 2004 ...... Visiting Scholar at Food animal Health

...... Research Program, OARDC/OSU

July 2004 to February 2005 ...... Contract at Food animal Health

...... Research Program, OARDC/OSU

April 2005 to November 2005 ...... Contract at the Istituto Zooprofilattico

...... Sperimentale Della Lombardia e Dell’Emilia

...... Romagna, Brescia, Italy

May 2006 to present ...... Graduate Research Associate, Veterinary

Preventive Medicine, The Ohio State

University

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Publications

1. Tibor Farkas, Brittney Fey, Edwin Hargitt III, Mark Parcells, Brian Ladman, Maria Murgia, Yehia Saif. Detection of novel picornaviruses in chickens and turkeys. Virus Gene, accepted for publication November 2011.

2. Abdul Rauf, Mahesh Khatri, Maria V. Murgia, Kwonil Jung and Yehia M. Saif. Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus. Veterinary Research (2011), 42(1):85.

3. Rauf A., Khatri M., Murgia M.V., Saif Y.M. Expression of perforin-granzyme pathway genes in the bursa of infectious bursal disease virus-infected chickens. Dev Comp Immunol (2011), 35(5): 620-627.

4. Tang, Y., Murgia, M.V., Ward, L., Saif, Y. M. Pathogenicity of turkey astroviruses in turkey embryos and poults. Avian Diseases (2006), 50(4): 526- 531.

5. Tang, Y., Murgia, M.V., Saif, Y. M. Molecular characterization of the capsid gene of two serotypes of turkey astroviruses. Avian Diseases (2005), 49(4): 514- 519.

Fields of Study

Major Field: Veterinary Preventive Medicine

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Table of Contents

STUDIES ON TURKEY PARVOVIRUSES ...... 1

DISSERTATION ...... 1

Abstract ...... ii

Acknowledgments ...... v

Vita ...... vii

Publications ...... viii

Fields of Study ...... viii

Table of Contents ...... ix

List of Tables ...... xii

List of Figures ...... xiii

Chapter 1: Literature Review of Animal Autonomously Replicating Parvoviruses .. 1

1.1 Etiology ...... 1

1.2 Epidemiology ...... 24

1.3 Pathogenesis ...... 27

1.4 Immunity ...... 30

ix

1.5 Treatment, Prevention and Control ...... 31

1.6 Diagnosis ...... 32

1.7 References ...... 36

Chapter 2: Prevalence of parvoviruses in commercial turkey flocks ...... 42

2.1 Summary ...... 42

2.2 Introduction ...... 43

2.3 Material and Methods ...... 44

2.4 Results ...... 47

2.5 Discussion ...... 49

2.7 Acknowledgements ...... 53

2.6 References ...... 54

Chapter 3: Detection of parvoviruses and other enteric viruses in commercial turkey flocks in four states of the United States between 2000 and 2010...... 61

3.1 Summary ...... 61

3.2 Introduction ...... 63

3.3 Material and Methods ...... 64

3.4 Results ...... 68

3.5 Discussions ...... 71

3.6 Acknowledgements ...... 75

x

3.7 References ...... 75

Chapter 4: Development of a Real-Time PCR for the detection of turkey parvoviruses...... 83

4.1 Summary ...... 83

4.2 Introduction ...... 85

4.3 Material and Methods ...... 86

4.4 Results and discussion ...... 89

4.5 Acknowledgements ...... 91

4.6 References ...... 91

Chapter 5: Experimental infections of SPF turkey poults with parvovirus and astrovirus...... 99

5.1 Summary ...... 99

5.2 Introduction ...... 100

5.3 Material and Methods ...... 102

5.4 Results ...... 106

5.5 Discussions ...... 109

5.6 Acknowledgements ...... 112

5.7 References ...... 113

Bibliography ...... 121

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List of Tables

Table 2.1. Information of turkey samples examined ...... 55

Table 2.2. TEM and PCR Results...... 56

Table 2.3 Correlation between TEM detection of SRV and PCR detection of parvoviruses

...... 57

Table 3.1 Correlation between transmission electron microscopy (TEM) and PCR/RT-

PCR ...... 76

Table 4.1 Information on turkey samples examined ...... 93

Table 4.2 Intra-assay Repeatability ...... 94

Table 4.3 Inter-assay Repeatability ...... 95

Table 4.4 Real-Time PCR vs. Conventional PCR detection of fecal samples...... 96

Table 5.1 Astrovirus RT-PCR results - Trial 1 ...... 115

Table 5.2 Astrovirus RT-PCR results - Trial 2 ...... 116

Table 5.3 Parvovirus Real-Time PCR results – Group 3- Trial 1 ...... 117

Table 5.4 Parvovirus Real-Time PCR results – Group 3- Trial 2 ...... 118

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List of Figures

Figure 1.1 Taxonomy of the family Parvoviridae ...... 2

Figure 2 MVM genome map and messenger RNAs...... 4

Figure 2.1 Electromicrographs ...... 58

Figure 2.2 Cladogram ...... 59

Figure 3.1 Overall prevalence of the tested enteric viruses ...... 78

Figure 3.2 Prevalence of the tested viruses by year ...... 79

Figure 3.3 Prevalence of the tested viruses by State ...... 80

Figure 3.4 Prevalence of the tested viruses by Age ...... 81

Figure 3.5 Prevalence of the tested viruses by health status of the flock ...... 82

Figure 4.1 Standard curve of pure plasmid copy number dilutions ...... 97

Figure 4.2 Standard curve of negative feces spiked with copy number of plasmid dilutions...... 98

Figure 5.1 Trial1- body weight trend assessed with linear equation ...... 119

Figure 5.2 Trial2- body weight trend assessed with linear equation ...... 120

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Chapter 1: Literature Review of Animal Autonomously Replicating

Parvoviruses

1.1 Etiology

1.1.1 Taxonomy

Parvoviruses belong to the family Parvoviridae (Figure 1.1) which includes two

subfamilies: Densovirinae, containing genera that infect invertebrates, and

Parvovirinae, containing genera that infect mammals and birds (ICTV, 2009).

Between 2008 and 2010, two new genera have been proposed in the

Parvovirinae subfamily: the hokovirus genus, which includes isolates of

porcine and bovine parvoviruses identified in Hong Kong (Lau et al., 2008); and

the cnvirus genus, which contains porcine parvovirus isolated in southeastern

China (Wang et al., 2010). Autonomously replicating parvoviruses belong to

four genera (parvovirus, erythrovirus, amdovirus, and bocavirus) within the

Parvovirinae subfamily.

1

Genus Densovirus Subfamily Iteravirus Densovirinae Brevidensovirus Pefudensovirus

Family Parvoviridae Genus Parvovirus

Erythrovirus Subfamily Dependovirus Parvovirinae Amdovirus Bocavirus Hokovirus Proposed ProposedGenera Cnvirus New Genera

Figure 1.1 Taxonomy of the family Parvoviridae

1.1.2 Morphology

Parvoviruses have an icosadeltahedral capsid structure. They are composed of

60 subunits made of two to three different proteins: VP1, VP2 and, depending on the species, the amino-terminal part of the latter can be cleaved, in the complete virion, into VP3 (Agbandje, 1995). The detailed structures of most of the parvoviruses are known at their atomic level via X-ray crystallography. One of the first structures to be uncovered was that of canine parvovirus (Luo, 1988).

The analysis of it confirmed the icosadeltahedral symmetry of the virion and

2 showed that each subunit was composed of an eight-strand anti-parallel β-barrel interconnected by a series of loops that make most of the surface of the virion.

The capsid has 5-fold, 3-fold and 2-fold rotational axes. Different structures were identified: a cylindrical structure created by the assemble of five anti- parallel β-strands at the 5-fold axes, a spike of 22 angstrom (Å) in length protruding from each of the 3-fold axes, a 15 Å “canyon-like depression” around each of the 5-fold axes, and a 15 Å “dimple-like depression” at the 2- fold axes between the spikes (Agbandje, 1995; Tsao et al., 1991).

1.1.3 Physicochemical Properties

Parvoviruses have a very stable icosahedral capsid. They are resistant to a wide range of pH, from acidic (pH 3) to basic (pH 9 up to 11.9 for Minute Virus of

Mice) as well as wet-heat, 80ºC for one hour. On the other hand, they are inactivated by wet-heat at 90 ºC for 10 minutes, formalin, β-propriolactone, hydroxylamine and oxidizing agents (Berns, 2007; Boschetti et al., 2003).

1.1.4 Genome Organization

Parvoviruses contain a linear single strand DNA (ssDNA) genome of about

5000 nucleotides. There is variability regarding which polarity of genome is packaged into the infective particles, for example: Minute Virus of Mice

(MVM) and H1 viruses package negative polarity genomes; on the other hand,

LuIII virus encapsidate negative or positive ssDNA with the same

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1 2 3 4 5 6 7 8 9 10 m u 0 0 0 0 0 0 0 0 0 0 MVM genome map

RNA-1 (4.8 kb)

RNA-2 (3.3 kb)

RNA-3 (3.0 kb)

Figure 1.2 MVM genome map and messenger RNAs (adapted from (Astell et

al., 1983)). The symbol ( ) indicates the splicing sites. The Symbol ( ) indicates the promoter’s start.

frequency as demonstrated by electrophoresis in non-denaturing conditions

(Bates et al., 1984). The genome structure is conserved among different species.

The first genome to be sequenced and studied in details is MVM, which is considered the parvovirus genus’ prototype. It has one hairpin loop at each end of the genome: 104 out of 115 nucleotide base-pair at the 3′-end; and 200 out of

206 nucleotide base-pair at the 5′-end. It contains two open reading frames; the nonstructural proteins are located at the left half of the genome while the structural proteins are at the right half. There are two promoters: one at 4.5 map units (mu) and the other one at 39 mu. These produce three transcripts, extending from the promoters to about 95 mu, that will be post-transcriptionally spliced (Figure 1.2), capped at the 5’-end and poly-A tailed at the 3’-end. The

4

RNA-1 (4.8 kilobases (Kb) in length) codes for the nonstructural protein; the

RNA-2 (3.3 kb) and RNA-3(3 Kb) code for the structural proteins, respectively,

VP1 and VP2 (Astell et al., 1983; Berns, 2007). In vitro studies of MVM- infected A-9 mouse cell line, showed the presence of a fourth RNA of 1.8 kb at low abundance (Pintel et al., 1983).

1.1.5 Viral Proteins

1.1.5.1 Structural Proteins

The genome of the autonomously replicating parvoviruses codes for two to four structural proteins depending on the strain. Bovine parvovirus (BPV), belonging to the bocavirus’ genus, has virion composed of four RNA-coded structural proteins, of 80 KDa,72 KDa, 65KDa and 60 KDa, as demonstrated by in vitro translation of mRNA extracted from infected bovine fetal lung cells and by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of purified virion (Lederman et al., 1983). On the other hand, rabbit parvovirus

(LPV), belonging to the parvovirus genus, has the virion composed of three

RNA-coded proteins of 96 KDa, 85 KDa and 75 KDa and a fourth protein of 70

KDa, which is the product of a proteolytic cleavage of the most abundant 75

KDa protein (Matsunaga and Matsuno, 1983). The other strains of the parvovirus genus have a virion composed of three proteins: two, VP1 and VP2, are produced by the translation of alternatively spliced mRNAs; and a third,

VP3, originated by the amino-terminal proteolitic cleavage of about 30 amino

5 acids of the VP2 within the mature virion (Cotmore, 1987; Tattersall et al.,

1977). The molecular weight of the three proteins is 80-86 KDa, 64-75 KDa,

60-62 KDa, for VP1, VP2 and VP3, respectively (Berns, 2007). Tattersall et al. analyzed, by SDS-PAGE, the protein composition of empty and mature virion of MVM, and found that the empty capsid contained only VP1 and VP2, while the mature virion contained all three proteins. They also calculated the number of proteins per virion, empty and mature, and found that VP1 is present at a fixed number in both types of particle, counting for 8-9 copies. On the other hand, the number of VP2 varies: in the empty virion it is present in 53-54 copies, while in the full virion the number of VP2 and VP3 depends on the virus preparation, counting 49 copies of VP2 and 4 of VP3 or 10 copies of VP2 and

47 of VP3. Moreover, they found that in MVM virus growth in some cell lines, such as RL5E, a fourth polypeptide of 50 KDa appears. The presence of this polypeptide did not influence the infectivity of the particle and it was considered a non-essential protein (Tattersall et al., 1976). Human parvovirus

B19, belonging to the erythrovirus genus, and Aleutian Mink Disease Virus

(AMDV), belonging to the amdovirus genus, have a capsid composed of two

RNA-coded proteins and, as for the other parvovirus strains, one make most of the capsid structure (Bloom, 1982; Cotmore et al., 1986).

The structural proteins are not glycosylated, but some are phosphorylated

(Berns, 2007). Fingerprinting analysis of trypsin and α-chymotrypsin treated empty and mature virions of MVM, demonstrated that the amino acid sequence

6 of VP2 is entirely contained within VP1, which contains an extra portion containing basic amino acids (Tattersall et al., 1977). This extra portion, of variable length depending on the strain, has been shown to contain nuclear localization signals (Tullis et al., 1993; Vihinen-Ranta et al., 1997), a DNA- binding domain (Willwand, 1988) and a catalytic and Ca++-binding domains of secretory phospholipase A2 (sPLA2) proteins (Zadori et al., 2001). The two motifs of sPLA2 are conserved among parvoviruses and in the majority of the cases are located inside the virion as demonstrated by the detection of the PLA activity in the alkali denatured and renatured capsid, but no in the intact one

(Zadori et al., 2001). The same group used porcine parvovirus to investigate the role of PLA2 activity in the life cycle of the virus. Their results showed that

PLA2 activity is important in the delivery of the viral DNA from the endosome/lysosome vesicles into the nucleus as demonstrated by site directed mutagenesis and in vitro infections studies (Zadori et al., 2001). The VP1 amino terminal extra portion (226 bases) of parvovirus B19 differs compared to the other autonomously replicating parvoviruses for its location and functions.

In fact, it is present on the surface of the virion as demonstrated by the immunoprecipitation of empty and mature virions with rabbit antisera raised against it. This location together with the fact that it contains a low amount of basic amino acids, lead to the hypothesis that it is not involved, as for the other parvoviruses, in the DNA binding. Moreover, it has been shown that this portion contains immunogenic epitopes and that the whole VP1 is involved in exposing

7 immunogenic epitopes of VP2 in capsids made of both types of proteins. The speculation is that this external part interacts at some point in the infection cycle with lipid membrane or that it forms the ligand for a membrane receptor

(Rosenfeld et al., 1992).

The VP2 has different functions in the life cycle of the virus: a. receptor-mediated entry of the virus into the permissive cells; b. capsid assembly and DNA packaging of virus progeny as demonstrated by

the fact that the in vitro transfection of MVM infectious clones containing

the nonstructural proteins together with VP2 or VP2 and an inactive VP1,

support DNA replication and capsid assembly of the viral progeny (Tullis et

al., 1993); c. it contains epitopes targeted by neutralizing antibodies.

1.1.5.2 Non Structural Proteins

The genome of autonomously replicating parvoviruses codes, in the majority of

the cases, for two nonstructural proteins, NS1 and NS2. The NS1 is a phosphoprotein of about 83KDa that plays important roles during viral

DNA replication and mRNA transcription. On the other hand, NS2 is a 25kDa protein that in MVM exists in three isoforms which differ in their carboxy terminal portions. They are localized in the cytoplasm where they are present as phosphorylated and nonphosphorylated proteins; the nonphosphorylated form is also present in the nucleus (Cotmore and Tattersall, 1990). The NS2 protein is

8 involved in capsid assembly (Cotmore et al., 1997), DNA replication (Naeger et al., 1990), mRNA translation (Naeger et al., 1993), and virion egress from the nucleus (Eichwald et al., 2002; Miller and Pintel, 2002). In order to determine the genomic coding sequences and the relationship between the nonstructural proteins, Cotmore and Tattersal performed immunoprecipitation studies using mice antisera raised against chimeric proteins produced by prokaryotic expression of plasmid containing three sequences of the left half of MVM genome. These sequences, designated A (from nucleotide 225 to 534), B (from nucleotide 1110 to 1638), and C (from nucleotide 2075 to 2291), correspond to the ORF 3 (A and B) and ORF 2 (C).

From those studies they found out that the two nonstructural proteins have the same amino-terminal region as demonstrated by the fact that antisera raised against the fragment A immunoprecipitate both proteins. However, the two proteins differ in the carboxy-terminal region, in fact, antisera raised against fragment B and C selectively immunoprecipitate NS1 and NS2, respectively

(Cotmore and Tattersall, 1986).

NS1 has several functions, some are virus-related and others are host-cell related. The former are: a. ATPase and helicase: these activities were demonstrated by in vitro ATPase

and helicase assays performed using a purified NS1 protein produced in Sf-9

cells via insertion of complete NS1 ORF into a baculovirus vector. The

helicase activity is ATP-dependent (Wilson et al., 1991).

9 b. Site-specific endonuclease: this activity is required for the resolution of the

DNA replicative form present as concatemer and for the subsequent

replication of the 5′-end and 3′-end of the newly synthesized viral DNA.

This was determined by in vitro studies in which a plasmid-cloned 5′ to 5′

junction of MVM replicative form, in the presence of NS1-containing cell-

free extract of HeLa cells, was resolved and new DNA synthesis completed

the ends (Cotmore et al., 1992). c. ATP-binding and DNA-binding: studies of immune isolation of DNA-

protein complexes, followed by gel electrophoresis, lead Cotmore et al. to

the discovery that NS1 specifically binds, in ATP-dependent manner, to the

3′-end origin of replication of MVM genome containing (ACCA)2 sequence

(Cotmore et al., 1995). Moreover, the fact that variation of those DNA

motives, present singly or as variably spaced repeated sequences, are

scattered throughout the viral genome and that NS1 binds to most of them,

suggested a chromatin-like role of the NS1 during genome replication

(Cotmore et al., 2007). An ATP-dependent activation of transcription of the

MVM promoter located at 38 m.u (P38) has been demonstrated by co-

immunoprecipitation and DNase I footprinting assays in presence or absence

of ATP (Christensen et al., 1995). d. Transcription activation: NS1 is involved in the MVM activation of the

transcription of mRNA from both promoters, at 4 m.u.(P4) and P38. This

activity is mediated via interaction with various cellular transcription

10 factors, through its carboxy-terminal portion. Among the transcription factors identified are:

i. SP1, a heterodimer responsible for the enhancement of the viral

transcription via interaction with NS1 as demonstrated by in vitro

protein retention studies in presence or absence of SP1. Moreover, co-

immunoprecipitaion of SP1-NS1 complexes with antibodies against

SP1 leaded to the hypothesis of a direct interaction between the two

proteins (Krady and Ward, 1995). This hypothesis was confirmed by in

vitro binding studies with GST-NS1 fusion protein (Lorson et al.,

1998)

ii. TFIIAα/β and TPB: in vitro binding studies with GST-NS1 fusion

protein demonstrated the interaction between these two transcription

factors and NS1 (Lorson et al., 1998)

iii. CREB binding protein (CPB): transient transfection assays using a

construct with luciferase gene under the control of P38 promoter

together with NS1 and various amount of CPB showed an increase in

the transcription of the luciferase proportional to the amount of CPB.

Moreover, the association of CPB-NS1 was demonstrated by co-

immunoprecipitation studies and it was localized in the C/H1 and C/H3

regions of CPB by pull-down assays. In addition, it was shown that this

interaction interferes with the transcription of p53gene (Ohshima et al.,

2001).

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The NS1 host-cell related functions are:

a. Interfering with the host cell cycle: parvoviruses have been demonstrated

to interfere with the cell cycle of the infected cells. Parvovirus B19 has

been shown to induce NS1-mediated cell arrest at the G1 phase and a

NS1-independent arrest at the G2/M phase of the cell cycle (Morita et al.,

2003; Morita et al., 2001). On the other hand, MVM has been shown to

arrest the cell cycle at S phase in p53-dependent but p21 and NS1-

indepent manner, and at G2 phase in p53 and p21-dependent manner and

that NS1 induces p21 but not p53 accumulation in G2 (Op De Beeck et

al., 2001).

b. Apoptosis: Parvovirus induces apoptosis in infected cells by activating

cellular apoptotic pathways. The role of NS1 in the activation of these

pathways has been investigated. The H1 virus induces apoptosis via

activation of caspase-3 pathways and it has been suggested that the

accumulation of NS1 is required to start the cleavage of the enzyme

PARP by the caspase-3 (Rayet et al., 1998). An apoptosis NS1-mediated

by activation of caspase-3 pathways has been suggested also for

parvovirus B19 infection in hepatocytes. Moreover, parvovirus B19

infection of hepatocytes activates the caspase-9 but not the caspase-8

pathways (Poole et al., 2004). Similar finding have been demonstrated in

MVM-infected PyF rat fibroblast cells, in fact, the apoptosis pathway of

infected cells was caspase-8 and p53 independent, but was dependent on

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the activation of caspase-3 and caspase-9 in NS1-mediated manner

(Cotmore, 1987).

1.1.6 Virus Life Cycle

1.1.6.1 Viral entry

Parvoviruses enter the target cells, for the most part, via receptor-mediated endocytosis, however macropinocytosis has also been demonstrated in case of

PPV when present in aggregates (Boisvert et al.; Parrish, 2010). Receptors and/or co-receptors have been identified for canine parvovirus (CPV), feline parvovirus (FPV), aleutian mink disease virus (ADV), mink enteritis virus

(MEV), minute virus of mice (MVM), human parvovirus B19 (B19), bovine parvovirus (BPV) and porcine parvovirus (PPV) (Boisvert et al.; Johnson et al.,

2004; Vihinen-Ranta et al., 2004). CPV and FPV are known to bind to the feline transferrin receptor-1 and in vitro infections studies showed that they can also bind to human transferring receptor expressed in TRVb-1 cell line leading to a productive infection (Parker et al., 2001). Moreover, both viruses can bind to the sialic acid in a pH-dependent manner, however, this binding is not necessary in order to have a productive infection as demonstrated by the fact that viral mutation impairing the receptor binding site did not affect the infectivity of the viruses (Barbis et al., 1992). In addition, CPV, but not FPV, has the ability to bind to the canine transferring receptor, and it has been shown that the mutation of two amino acids, at position Lys93Asn and Asp323Asn of VP2 in FPV, is

13 responsible for this property (Hueffer et al., 2003). MEV has been shown to use the tranferrin receptor in in vitro infection studies in Crandell Feline Kidney cells (CRFK) in which the reduction of the expression of this receptor caused the block of MEV infection (Park et al., 2005). ADV binds to a CRFK cell membrane receptor identified by overlay protein binding assay in which a mixture of membrane proteins derived from CRFK cells were blotted into a membrane and incubated with labeled VP2 empty capsids. This receptor, named

ADV binding protein (ABP), has a moleculr weight of 67KDa (Fox and Bloom,

1999). MVM strains recognize sialic acids α2-3- linked to multiple Gal-GlcNAc motives as established by glycan array screening. Moreover, MVM immunosuppressive and MVM prototype mutant K638R also recognize sialic acid α2-8- linked to multiglycans (Nam et al., 2006). Two other viruses use sialic acid as a receptor for viral entry, BPV and PPV. BPV was found to bind sialic acids α2-3-N or O-linked as demonstrated by in vitro binding studies using various cell lines and a combination of sialidases and sialyltransferase

(Johnson et al., 2004). On the other hand, the use of a neuraminidase inhibitor allowed the identification of sialic acids as the cellular receptor for PPV

(Boisvert et al.). Human parvovirus B19 binds to the globoside P antigen of erythrocytes as demonstrated by the fact that a purified lipidic fraction containing ceramide oligohexosides (globoside) inhibited the virus-mediated hemagglutination of human erythrocytes. Moreover, they tested human blood with different P phenotypes and showed that B19 could agglutinate only

14 erythrocytes belonging to the P phenotype (Brown et al., 1993). Recent binding studies, employing a recombinant human parvovirus B19 vector, re-evaluated the role of P antigen in the viral entry of the virus and proved that P antigen is necessary and sufficient for the viral absorption to the target cell, but it requires a co-receptor for its cell entry. This conclusion was drawn by the observation that B19 was able to attach to the P antigen but not to enter into K565 and HL-

60 hematopoietic cell lines (Weigel-Kelley et al., 2001). Subsequent studies identified the integrin α5β1 as co-receptor for B19 cellular entry (Weigel-Kelley et al., 2003).

1.1.6.2 Viral trafficking from the cytoplasm to the nucleus

The internalized virus follows the endosomal pathway to reach into the proximity of the nucleus. Parker et al. demonstrated that CPV-transferrin receptor internalization was dependent on dynamin as demonstrated by the slow movement of vesicles-containing virus to perinuclear locations (Parker and

Parrish, 2000). The CPV pathway of infection in NFLK cell line was investigated by fixing the cell at various times post infection (p.i.) and using immunofluorescent microscopy with antibodies against capsid and NS1 proteins. When anti-capsid antibodies were employed, CPV-containing vesicles were found at the periphery of the cell at 30 minutes p.i., at perinuclear location at 3 hour p.i. and inside the nucleus after 10 hours p.i. However, the use of anti-

NS1 antibodies allowed the identification of NS1 inside the nucleus at 8 hours

15 p.i. In addition, the virus was detected, by immune electron microscopy, in late endosomes at 1 hour p.i. and in lysosomes at 3 hours p.i. The vesicles- containing viruses were found to be associated with microtubules and this association was found to be essential for their trafficking to the nucleus as demonstrated by their localization at the periphery of the cell at 12 hours p.i. in nocodazole-treated cells. Moreover, antibody-mediated inhibition of the activity of dynein had the same effect as nocodazole in the localization of the vesicles- containing viruses at the periphery of the cell indicating the involvement of this protein in their transport to the nuclear periphery (Suikkanen et al., 2002). The

CPV must to go through the endosomic pathway in order to give rise to a productive infection as demonstrated by in vitro studies in which CPV microinjected directly into the cytoplasm was not able to replicate (Vihinen-

Ranta et al., 1998). Furthermore, the requirement for an acidic endosomal environment is a condition necessary for viral replication as shown by the fact that pre-treatment of cells with lysosomotropic compounds, raising the endosomal intracellular pH, impaired viral replication (Basak and Turner,

1992). It has also been demonstrated that, in the endosomal vesicles, MVM virions undergo to three simultaneous capsid modifications: externalization of

N-terminus of VP1, cleavage of VP2 N-terminus, uncoating of the viral genome. These capsid changes are pH-dependent as shown by the fact that they were blocked in A9 cells infected with MVM in the presence of a drug, chloquine, that raise the internal endosomal pH (Mani et al., 2006). The acidic

16 environment of the endosomes vesicles is a condition necessary but not sufficient for a productive infection as shown by the fact that acidic pretreatment of CPV previa microinjection did not enable viral replication

(Vihinen-Ranta et al., 1998). Once virions are in perinuclear locations, are released via PLA2-mediated activity as demonstrated by the fact that MVM virions containing PLA2 mutated in the active domain, are not released from the endosomal vesicles (Farr et al., 2005). A PLA2-mediated mechanism has been demonstrated also for CPV in experiments in which inhibitors of PLA2 leaded to a nonproductive infection. The proposed mechanism of vesicles escape is not via rupture of the lysosomal membrane, but it seems that the virus permeabilize the membrane as demonstrated by experiments of co-internalization of viruses together with various markers in which a size-dependent virus-mediated release of the markers is detected (Suikkanen et al., 2003b).

The nuclear entry of the parvovirus it not well characterized. It is hypothesized that parvoviruses, released from the endosomal vesicles, enter the nuclear pores, which have a diameter of about 25 nm, through the nuclear signal sequences present at the N-terminus of VP1, that were externalized within the endosomal vesicles (Suikkanen et al., 2003a).

1.1.6.3 Viral DNA replication

It is not clear if the DNA is released from a disassembled capsid or if externalized sequences at the 3′-end of the genome can be used for replication.

17

The method of parvovirus replication is thought to be via “rolling hairpin amplification” (Berns, 2007):

1. the hairpin loop at the 3′-end of the genome is used as primer for the

synthesis of the complementary strand by the cellular DNA polymerase

2. the polymerase continues the synthesis until it reaches the hairpin loop at

the 5′-end where it stops

3. the newly synthesized and the template filament are covalently linked at

that point

4. the protein NS1 makes a nick at the 3′-end of the newly synthesized

filament, 18 nucleotides downstream from the 5′-end of the parental

strand

5. NS1 is covalently linked to the newly formed 5′-end, while the 3′-end is

used to fill in the gaps in the new filament

6. Denaturation at the right end of the duplex, formed by the parental and

newly synthesized filaments, lead to a hairpin loop configuration and to a

new DNA synthesis from the free 3′-end through the all duplex DNA

including the parental strand

7. The result of this new replication is the formation of a tetramer structure.

The tetramer is composed by three newly replicated filaments plus the template filaments; these filaments have left-end-left end and right-end-right end orientation. This structure is resolved by single genome excision and replication of terminal sequences with mechanisms called” terminal resolution” and

18

“junction resolution” for the right-end and left end, respectively (Cotmore,

1995). The terminal resolution mechanism involves the binding, as dimer, of the

NS1 protein to its DNA target sequence “ACCA”, followed by a nick within a specific DNA sequence “CTWWTCA”. The nick leave a 5′-end bound to NS1 and a free 3′-end available for the polymerase to synthesize the other filament’s end (Cotmore, 1995). On the other hand, the junction resolution involves the endonuclease activity of NS1, but in this case, the nick of the DNA in the target site “GA/TC” (“bubble” of the left-end hairpin loop), release two different structures: a linear “extended form” and a hairpin loop form, called “turnaround form” (Cotmore, 1995).

1.1.6.4 Transcription

The MVM genome has two promoters, P4 and P38, which direct the transcription of the ORFs coding for the non-structural proteins and the structural proteins, respectively. The parvovirus genome has two introns, one large, between position 10 and 39 m.u.in MVM, and one small, between 44 and

46 m.u. in MVM (Astell et al., 1983). The two mRNAs transcribed from the P4 promoter contain both introns, but the splicing pattern is different, in fact, the mRNA coding for NS1 has only the small intron spliced out, on the other hand, the mRNA coding for NS2, has both introns spliced out. The mRNA transcribed from the MVM P38 promoter give rise to the VP1mRNA, when the small intron is removed using the donor site at position 2317 and the acceptor site at position

19

2399, or VP2 mRNA, when the small intron is removed using the donor site at position 2280 and an acceptor site at position 2377 (Jongeneel et al., 1986;

Morgan and Ward, 1986).

The transcription from the two promoters occurs at different times with the mRNAs from the P4 promoter transcribed earlier than the one from the P38 promoter as demonstrated by RNase protection assays in double blocked synchronized A9 murine cell line infected with MVM (Clemens and Pintel,

1988). Different cellular factors together with the NS1 protein are responsible for the initiation of the transcription. The transcription from the P4 promoter is activated as soon as the cell enters the S phase and depends on the E2F transcription factor binding to its DNA binding site located in the proximity of

P4 as shown by in vitro studies in which a MVM infectious clone with mutated

E2F binding site was unable to replicate unless NS1 protein was added (Deleu et al., 1999). Two other proteins, CREB and Sp1, have DNA binding sites upstream P4 promoter and are involved in its activation (Ahn et al., 1989;

Perros et al., 1995). The promoter P38, transcribed later during infection, has various upstream regulatory elements: a TATA box, a GC box, the so called

“transactivation response region” (TAR) and NS1 binding sites. In vitro mutagenesis studies have shown that NS1 is necessary for P38 transcription and that the only elements necessary for its action were the TATA box and the GC box together with its DNA binding sites. The TAR, which was shown to contain a NS1 binding site, was not essential in studies where a full MVM genome was

20 employed, however, when partial genome sequences without NS1 binding sites were used it became indispensable (Lorson et al., 1996).

1.1.6.5 Translation

The translation of parvovirus proteins occur in the cytoplasm using the cellular machinery and follows the order of production of the corresponding mRNA.

The first proteins produced are NS1 and NS2 that are post-translational phosphorylated (Berns, 2007), on the other hand the structural proteins are produced later during infection and are assembled into trimers before their translocation within the nucleus where they are going to be assembled into capsids (Riolobos et al., 2006).

1.1.6.6 Capsid assembly and DNA packaging

Parvovirus capsid assembly occurs within the nucleus and the process has been studied in details for the MVM parvovirus. It has been shown to require various steps: trimerization of capsid proteins in the cytoplasm, translocation of the trimers into the nucleus, and within the nucleus, trimers rearrangement to be assembled into capsids (Bloom, 1982). The trimerization of VP proteins in the cytoplasm, as necessary step for the translocation of the VP proteins into the nucleus, was first suggested by Lombardo et al. by in vitro studies of transfection of NB324K cell line with plasmid containing MVM immunosuppressive (MVMi) strain’s genome with various mutations in the VP

21 gene. They showed that the nuclear translocation of VP1was not affected by any of the mutations, suggesting the implication of the NLS in the unique N- terminal region; on the other hand all mutations impaired the translocation of

VP2, suggesting an involvement of a specific protein folding. Moreover, VP1 interacted with the translocation- impaired VP2 in the cytoplasm and helped its translocation into the nucleus. They also identified a conserved motif rich in basic amino acids in the β-strand I of VP2 which is a nuclear localization motif

(NLM) and it is essential for the translocation of VP2 into the nucleus as demonstrated by site-directed mutagenensis studies in which double mutants in the NLM region were retained in the cytoplasm. In addition, a trimeric assembly of VP1/VP2 was supported by the following: VP1/VP2 ratio in the cytoplasm is known to be of 1:5, VP1 is able to interact and transport into the nucleus about

50% of the translocation- impaired VP2, and a trimer conformation would allow the external exposure of the β-strand I of VP2 with its NLM (Lombardo et al., 2000). The trimers formation was confirmed in subsequent studies via

DMS cross-linking followed by SDS-PAGE, that identified two types of trimers: one composed of only VP2s and one composed of VP1 and VP2 at 1:2 ratios. Moreover, transfection of an infectious plasmid containing a double mutation in the NLM region caused the cytoplasmic accumulation of the two type of trimers (Riolobos et al., 2006). Once the trimers are translocated into the nucleus they undergo rearrangements creating hydrophobic protein-protein interactions. In order to investigate the intertrimers interaction, various residues

22 were selected for mutations based on the following characteristics: amino acids were conserved among parvoviruses; their side chain made with few intramonomer interactions, and they did not have any intertrimer interactions.

None of the mutations impaired the formation of the trimers or their nuclear translocation; however, 14 out of 28 mutants were not able to be assembled into capsids. The mutated residues were spread over the interface of the monomers composing the trimers and were the ones involved in the strongest protein- protein interactions (Bloom, 1982).

The packaging of the DNA occurs in a preformed capsid and it seems to involve the channel at one of the 5-fold axes as portal entry as demonstrated by transfection studies of MVM with a leucine to tryptophan mutation at position

172 in the VP gene which block the channel and impair the DNA packaging process (Willwand, 1988).

1.1.6.7 Viral egress

The MVM parvovirus progeny virions exit from the nucleus via the interaction of NS2 with the nuclear receptor Crm1 (involved in the active transport of proteins into the cytoplasm) as demonstrated by in vitro transfection studies in which NS2 protein residues implicated in the Crm1 interaction were mutated.

This mutant transfected into A9 cells showed a remarkable decrease in production of newly synthesized ssDNA and an impairment of progeny virons nuclear egress (Miller and Pintel, 2002). Once virions are in the cytoplasm,

23 another protein, gelsolin, was suggested to be involved in their cellular egress through lysosme or late endosome vesicles. In fact, after infection gelsolin was localized close to the plasma membrane and around the nuclear membrane; moreover, viral progeny were detected within vesicles only in the presence of gelsolin (Bar et al., 2008).

1.2 Epidemiology

1.2.1 Natural Hosts

Serological surveys together with experimental infections have identified the natural hosts for the autonomously replicating parvoviruses and determined that the majority of the viruses have one specific host (Hirt, 2000). Nonetheless, some parvoviruses have broader host ranges which are: Felides for feline panleukopenia virus (FPV) , Canides for canine parvovirus type-2 (CPV-2),

Felides and Canides for CPV-2 variants (Steinel et al., 2001). In a recent study,

Allison et al., identified a variant of CPV-2 infecting raccoon and phylogenetic analysis of VP2 gene showed that it was an intermediate host variant between

CPV-2 and CPV-2a (Cotmore, 1995).

1.2.2 Transmission

1.2.2.1 Horizontal Transmission

Autonomously replicating parvoviruses are generally excreted at high titer in feces, urines, saliva, mucus, and moreover are very resistant in the environment, 24 therefore the main route of transmission is considered through direct contact with infected excretes, contaminated objects, environment or mechanical transfer (Hirt, 2000; Steinel et al., 2001). The persistence of parvovirus in the infected host as well as its shedding with the body fluids has been investigated in various experimental and natural infections. It has been shown that fecal shedding of MVMi in oronasally infected mice could be detected as early as 1 day post infection (dpi) but drastically declined after 20 dpi; on the other hand the virus persisted in the spleen up to 30 dpi (Janus et al., 2008). Intranasal inoculation with parvovirus B19 of healthy volunteers showed viremia with concomitantly viral shedding in nasal washes and gargle samples at 7 and 11 dpi (Anderson et al., 1985). The shedding of ADV in asymptomatic but persistently infected ferrets, was demonstrated in blood, urine, feces tested by

PCR over a 2 year span (Pennick et al., 2005). An epidemic of parvovirus B19 was used, in immunocompromised patients, to determine the shedding of the virus in blood. It was shown that blood was positive by nested PCR from 2 to 6 months and in one case a patient with chronic arthropathy became persistently infected and it was possible to detect the virus up to one year (Musiani et al.,

1995).

1.2.2.2 Vertical transmission

The vertical transmission has been demonstrated for rat parvovirus, PPV,

MVM, ADV, BPV, FPV, CPV parvoviruses (Hirt, 2000; Jordan and Sever,

25

1994). Kilham and Margolis (1996) isolated a strain of rat virus, called Sp strain, from pregnant rats. The naturally infected rats had resorption of the majority of the fetuses and the newborn were weak and with jaundice. The experimental infection of newborn hamsters showed one of the typical pathology of parvovirus infections in rats, cerebellar ataxia. Moreover, the experimental infection of pregnant rats with the same isolate allowed:

- the reproduction of the disease;

- the confirmation of the transplacental infection based on the detection of

higher titer of viruses in the fetus compared to the mother;

- the identification of another route of transmission: the milk from the

mammary gland. In fact, milk collected from the stomach of the newborn

rat had a high titer of viruses (Kilham, 1966).

Parvovirus B19 was detected in tissues of fetuses with hydrops fetalis by

radioimmunoassay and electron microscopy (Clewley et al., 1987).

1.2.3 Mortality and Morbidity

Parvovirus infections could be lethal in case of in utero infections or newborn animals. The outcome of intrauterine infections for all parvoviruses depends on the stage of pregnancy in which the infection occurs and on the immune status of the mother. In fact, it has been shown that B19 infections within the first trimester of pregnancy determine abortion; in the second trimester hydrop fetalis and at termini stillbirth (Jordan and Sever, 1994). Moreover, it was

26 reported that mothers infected with parvovirus B19 that developed a sufficient antibody response gave birth to healthy babies (Brown and Ritchie, 1985;

Jordan and Sever, 1994).

The morbidity in parvovirus infections is high for FPV, CPV, and BPV which are known to be excreted in the feces at high titer and maintain their infectivity in the environment for long time (Kahn, 1978; Manteufel and Truyen, 2008).

1.2.4 World distribution

The majority of the species of parvoviruses are distributed worldwide, such as

FPV, CPV-2 variants, B19, BPV (Berns, 2007; Manteufel and Truyen, 2008).

On the other hand, CPV-1 distribution is less uniform among different countries

(Manteufel and Truyen, 2008).

1.3 Pathogenesis

1.3.1. Fetuses, Neonates, and young animals

Parvoviruses require actively replicating cells for their infection therefore their main targets are fetuses, neonates and young animals. Different types of pathologies have been identified in parvovirus infections of young animals:

- FPV: cerebellar hypoplasia and hydrocephalus, retina dysplasia (Lamm and

Rezabek, 2008)

- CPV: myocarditis is the main pathological finding (Lamm and Rezabek,

2008) 27

- ADV: interstitial pneumonia has been found in naturally and experimentally

infected mink (Alexandersen et al., 1994; Bidin M, 2011)

- BPV: diarrhea, vomiting, myocarditis, respiratory disease in naturally and

experimentally infected calves (Durham et al., 1985; Jordan and Sever,

1994).

1.3.2. Adults animals and asymptomatic carriers

The majority of adult animals are refractory to parvovirus infection or asymptomatic carriers. However, there are some examples of productive infection in adults: feline panleukopenia virus, mink enteritis virus, canine parvovirus, aleutian disease virus and some strains of rat parvoviruses (Coleman et al., 1983; Mase M, 2009). FPV infections in adults is mainly characterized by panleukopenia, while in CPV infections a moderate lymphopenia has been described, both viruses affect the intestinal cells leading to enteritis (Parrish,

1995). Adult aleutian mink infected by ADV showed sign of plasmacytosis, hyperglobulinemia, immune-complex-mediated glomerulonephritis and arteritis

(Best and Bloom, 2005). Infection with MVM without clinical signs occurs in adults mice (Jordan and Sever, 1994).

1.3.3. Pathogenesis’ factors

Parvovirus infections give rise to a different array of syndromes and pathologies or can just be clinically asymptomatic. Various factors play a role in the

28 outcome of the infection. These can be grouped into three categories: virus, host and cellular related factors. The virus factors are associated with the genetic variability of the genome due to mutations. It has been observed that in vitro passage of parvoviruses resulted in a reduction in the pathogenesis of the isolate. On the other hand, the passage of the virus in vivo resulted in either no variation or increase in pathogenicity compared to the original isolate.

Mutations could affect different functions such as organ tropism, ability and/or efficiency of viral replication, types of viral particles produced, complete and infectious vs. empty or defective interfering particles.

The host related factors are genetic background and age of the host, and

prevalence of parvovirus antibodies in the host population.

The cellular related factors can give rise to two different scenarios:

restrictive infections, and abortive infections, as supported by in vitro studies.

An example of restrictive infection is given by the in vitro host range of the two allotropic variants of Minute Virus of Mice (MVM), the so called prototype

(MVMp) and immunosuppressive (MVMi). These variants are antigenically closely related as demonstrated by virus neutralization test with rabbit hyperimmune antiserum raised against MVMp that neutralize both variants, but do not react with another rat parvovirus, H-1 (Tattersall and Bratton, 1983).

The MVMp and MVMi have a restrictive tropism for, respectively, mouse fibroblasts A9 and mouse T-lymphocytes S49 cell lines, however both can give

29

a productive infection in a hybrid cell line of mouse fibroblasts and T-

lymphocyte (Mase M, 2009; Tattersall and Bratton, 1983) .

1.4 Immunity

The immunity plays an important role in the pathogenesis of most parvovirus infections. The immune response against parvoviruses is both humoral and cellular-mediated; however, the former plays a major role in the pathogenesis of parvovirus infections. The production of anti-virus antibodies could be protective, as in the case of B19 parvovirus infections, or could exacerbate the pathogenesis of the infection, as in the case of ADV infection in adult aleutian mink (Best and Bloom, 2005). In fact, infections with parvovirus B19 determine the production of neutralizing antibodies starting from the second week post- infection with virus-specific IgM production, lasting for a week, and being then substituted by IgG as demonstrated by experimental infection of human volunteers (Anderson et al., 1985). Moreover, the immune response has been shown to confer protection from successive infections as demonstrated by the protection conferred by immunoglobulin therapy in the clearance of the virus in persistently infected patients (Brown et al., 1994; Kerr et al., 2002). However,

IgM form immune complexes with the virus and it is suggested that they are the cause of some of the pathology observed during infections such as skin rash

(Anderson et al., 1985).

30

On the other hand in ADV infected adult Aleutian minks, the antibody response it is not protective, but determine the formation of circulating virus-antibody complexes that cause the observed pathology (Best and Bloom, 2005).

1.5 Treatment, Prevention and Control

There are no specific treatments for the majority of the animal parvoviruses infections, the only one is supportive care based on the symptoms. On the other hand, immunoglobulin therapy for parvovirus B19 infections has been successfully used in cases of chronic fatigue syndrome (Kerr et al., 2003), and other parvovivurses persistent infections (Berns, 2007; Lamm and Rezabek,

2008).

The prevention of parvovirus infection in carnivores can be achieved by the use of vaccines. Inactivated vaccines against FPV, mink enteritis virus (MEV),

CPV-2 and CPV-2b have been produced and have been shown to give a short life protection in domestic cats and dogs, however they are recommended for their safety (Steinel et al., 2001). Modified live vaccines are also available against the same parvoviruses, and they induce a better protection even in presence of maternally derived antibodies (Squires, 2003), however, there is also some concern for the release of a live virus in the environment that could cause disease in non target species (Steinel et al., 2001).

31

1.6 Diagnosis

Different diagnostic methods have been developed for the diagnosis of parvoviruses. Serologic methods were used since the first identification of parvovirus in various species to determine their prevalence and the identification of their natural hosts (Hirt, 2000). In addition, antigen-detection methods and molecular methods such as PCR and Real-Time PCR are nowadays widely used for the rapid detection of animal and human parvoviruses (Jordan, 2001; Squires, 2003).

1.7 Current knowledge on poultry parvoviruses

1.7.1 Genome organization

The genome sequence of chicken and turkey parvoviruses has been recently obtained (Day and Zsak, 2010). The full length genome of the chicken ChPV

ABU-P1 strain is 5257 bases, while the one of turkey TuPV1078 strain is 4642 bases. The 3′ and 5′-end of the ABU-P1 genome contains 39 inverted repeated bases within 206 bases direct sequences that are identical at both ends. The fact that the direct repeated sequences at the 3′ and 5′-end are identical is a tract that is in common with human parvovirus B19 even though the length of those sequences in the latter is longer (Deiss et al., 1990). The overall structure of the genome of poultry parvoviruses is common with the other parvoviruses. It has two large open reading frames (ORF) which code for NS1 protein and VPs

32 proteins. However, in the chicken genome, there is also a small ORF between the previous two that potentially codes for a protein (NP1) of 101 amino acids of unknown functions. The turkey TuPV1078 has the same structure of the chicken regarding the NS1 and VPs proteins and the NP1 protein, however has one extra ORF coding for an NP protein of 69 amino acids (Day and Zsak,

2010).

1.7.2 Viral Proteins

The non-structural protein NS1 is composed of 694 and 627 amino acids in chicken ABU-P1 strain and turkey TuPV1078 strain, respectively; and it contains a conserved P-loop motif. It shares between 45.2 and 53.9 % of amino acids similarity with the other parvoviruses; and 89.3 to 100% when comparing chicken ABU-P1strain and turkey strains TuPV260 and TuPV1078 (Day and

Zsak, 2010).

There are three predicted structural proteins (VP1, VP2 and VP3) coded in the

ORF at the right half of the genome. VP1 is composed of 675 amino acids in the chicken ABU-P1 strain and 667 amino acids in the turkey strain TuPV1078.

VP1 of chicken ABU-P1 shares 95.1% and 100 % similarity at the amino acids level with TuPV1078 and TuPV260 turkey strains, respectively. However, its amino acids similarity with the other parvovirus is lower, counting between

46.6 % and 59%. The VP2 is composed of 536 and 535 amino acids in chicken

ABU-P1 and turkey TuPV1078 strains, respectively (Day and Zsak, 2010).

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1.7.3 Pathogenesis

Parvovirus in poultry has been identified in cases of enteritis, stunting syndrome, but also in healthy flocks (Bidin M, 2011; Kisary, 1984; Palade,

2011a, b; Trampel et al., 1983; Zsak et al., 2009; Zsak et al., 2008).

Nonetheless, their role in the enteric syndromes of poultry has not yet been elucidated. Recently parvoviruses have been associated in a case of cerebellar hypoplasia and hydrocephalus in 1-day-old chickens (Marusak et al., 2010).

Experimental infection studies with a chicken origin parvovirus was performed using 1-day-old SPF white leghorn chicken and commercial broiler chickens

(Kisary, 1985a). The researchers were able to reproduce the stunting in the broiler chickens, in fact the body weight gain of the infected chickens was 40% less compared to the controls. On the other hand, there was no effect in weight loss in the SPF white leghorn chickens (Kisary, 1985a). However, another group did not succeeded in the reproduction of the stunting in commercial broilers chickens using the same isolate as Kisary (Decaesstecker et al., 1986).

1.7.4 World distribution

Parvovirus is widely distributed in the US as demonstrated by a survey in which samples from 54 chicken farms in 7 US states and 29 turkey farms in 5 US states were tested. Those samples were collected from birds of less than 2- weeks of age and between 2 and 7 weeks of age. The researchers found a

34 prevalence of 77 % and 78% in chickens and turkeys, respectively (Zsak et al.,

2009). In Hungary, poultry parvoviruses were identified in chicken and turkey flocks as well as in healthy chicken flocks (Palade, 2011b). Moreover, turkey parvoviruses have been identified in cases of poultry enteritis complex (PEC) and poultry enteritis and mortality syndrome (PEMS) in 46.9% of turkey flocks composed of turkeys of less than 7 weeks old (Palade, 2011a). Furthermore, in a recent survey of Croatian chicken and turkey flocks affected by enteritis, parvoviruses have been identified in 7out of 9 chicken flocks and in 1out of 6 turkey flocks (Bidin M, 2011).

1.7.5 Diagnosis

In the 1980’s the transmission electron microscope was the diagnostic method that allowed the identification of turkey and chicken parvoviruses in diseased birds (Kisary, 1984; Trampel et al., 1983). An indirect immunofluorescent test was also developed for the detection of parvovirus in broiler chickens (Kisary,

1985b). In 2009 Zsak et al. developed a PCR test targeting the NS protein of chicken and turkey parvoviruses (Zsak et al., 2009). This test has been currently used in the recent prevalence studies in the US and in Europe (Bidin M, 2011;

Palade, 2011a, b; Zsak et al., 2009). An Enzyme-Linked-Immunosorbent-Assay

(ELISA) test for the detection of chicken parvovirus antibodies was also developed (Strother and Zsak, 2009). This test was validated in experimental

35

infected poults; however, it was not employed for any serologic surveillance

study.

1.8 References

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Chapter 2: Prevalence of parvoviruses in commercial turkey flocks

2.1 Summary

Turkey parvovirus belongs to the family Parvoviridae, subfamily Parvovirinae, genus parvovirus. Since the initial report on turkey parvovirus in the United States in 1983, there have been no further reports of parvovirus in turkeys until 2008. The aims of our study were: to determine the prevalence of parvovirus in commercial turkey flocks using

PCR; to determine their genetic relationship to previous strains identified in North

America and Europe; and to test samples for enteric viruses by transmission electron microscopy (TEM). A total of 169 fecal samples collected from 42 turkey farms in four different states in the U.S. between 2000 and 2010 were examined. We found that the most detected viruses by TEM were small round viruses accounting for 52% of the samples examined; on the other hand, the PCR detected parvoviruses in 71% of the samples. The phylogenetic analysis of partial NS gene sequences showed a certain degree of variability among the turkey samples tested in the study. Moreover, there was a clear dichotomy in the phylogenetic tree between chicken and turkey samples, with the exception of one turkey isolate of 2000 which clustered together with the chicken group.

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2.2 Introduction

Parvoviruses belong to the Family Parvoviridae, which is divided into two subfamilies:

Densovirinae and Parvovirinae. Chicken and Turkey parvoviruses belong to the genus parvovirus of the Parvovirinae subfamily . Parvovirus has an icosahedral capsid of approximately 18 to 26 nm in diameter and contains a single strand DNA of negative polarity of 4-6 kb in length (Berns, 2007)

In 1983, Trampel et al. reported for the first time the presence of parvoviruses in turkeys affected by stunting, enteritis and high mortality (Trampel et al., 1983). Analysis of a thin section of ileum by EM permitted the identification of hexagonal particles of 15-19 nm in diameter within a nuclear inclusion body. The following year, Kisary et al. in Hungary identified by EM, parvovirus particles in feces of chickens with stunting syndrome

(Kisary, 1984). The following years, their findings were confirmed by determining that its genome was composed of a single stranded DNA of about 5.2 Kb in size and the DNA self replicated in vitro. In addition, the virus was able to replicate in chickens and chicken embryos (Kisary, 1985a; Kisary et al., 1985). There have been no further reports of parvovirus in chickens or turkeys until 2008 when Zsak et al. determined the partial sequence of chicken and turkey parvoviruses by random PCR followed by sequence analysis (Zsak et al., 2008). Since then, the same group has developed a PCR test with primers targeting the non structural (NS) gene of the chicken and turkey parvoviruses

(Zsak et al., 2009), and later the complete genome sequence of one chicken and two turkey parvovirus strains was also determined (Day and Zsak, 2010). Zsak et al. used the above mentioned PCR to test samples collected in different years from commercial 43 chicken and turkey flocks in various US states. They found a parvovirus prevalence of

77% and 78% in chicken and turkeys flocks, respectively (Zsak et al., 2009). This report shows a widespread distribution of parvovirus, although it does not provide any information on the health status of the flocks from which the samples were collected. In

2010, Marusak et al. reported on parvovirus-associated cerebellar hypoplasia and hydrocephalus in broiler chickens (Marusak et al., 2010) and the following year Palade et al. reported the presence of Parvoviruses, using Immunohistochemistry, TEM and PCR, in Hungarian broiler flocks affected by enteric diseases; and by PCR in two healthy broiler flocks (Palade, 2011b).

To better understand the parvovirus epidemiology in turkeys, fecal samples collected from 42 turkey farms in four different states in the US between 2000 and 2010 were analyzed: we specifically determine the prevalence of parvovirus in commercial turkey flocks using PCR, their genetic relationship to previously identified strains of North

America and Europe, and the presence of other enteric viruses by TEM.

2.3 Material and Methods

Samples

Samples of feces, litter or intestinal contents were collected from Virginia (14 farms),

North Carolina (14 farms), Ohio (3 farms), and Pennsylvania (11 farms) during the years

2000 to 2010. The age of the birds was ranged from two to nineteen weeks. Information was not available regarding the health of the flocks for the samples collected in 2000 and

2005. The rest of the samples were from flocks affected by stunting syndrome, enteritis, 44 and increased mortality (Table 2.1). The mortality, in certain cases, was 0.15%-1.7% per day, in other 19% until the day of samples collection, or it was indicated as “increased mortality”. The samples were diluted 1:10 in 0.01M Phosphate Buffer Saline (PBS) pH

7.4, clarified at low speed centrifugation and filtered through 0.45 µm syringe filter. The filtrates were then used for TEM and for DNA extraction.

Transmission Electron Microscopy

For TEM analysis, 200 µl of filtered samples were subjected to ultracentrifugation through 20% (w/w) sucrose cushion in Airfuge ultracentrifuge (Beckman Coulter Inc.,

Fullerton, CA) at 30 psi for 15 minutes. The supernatant was discarded and the pellet was resuspended into 400 µl of double distilled water filtered through 0.22 µm syringe filter.

The sample was then ultracentrifuged at 30 psi for 15 minutes. The supernatant was discarded and the pellet was stained with a solution of 3% Phosphotungsic Acid-0.4% sucrose. It was then applied to a 300 mesh formvar coated copper grid that was analyzed by Hitachi 7500 Transmission Electron Microscope.

DNA extraction

Fifty microliters of the filtered samples were subjected to nucleic acid extraction in

MagMAXTM Express Magnetic Particles Processor (Applied Biosystems, Foster City,

CA) by using MagMAXTM-96 Viral RNA Isolation Kit (Ambion/AB, Austin, TX) following the manufacturer’s instructions.

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Conventional PCR for the detection of turkey parvoviruses

The PCR was performed using published primers, PVF1 and PVR1 (Zsak et al., 2009) and a PCR Master Mix (Promega, Madison, WI). The primers target a conserved region in the NS gene and the PCR amplifies a 561 bp product. The reaction volume was 25 µl with 1µM of each primer. The PCR cycle was: denaturation at 95°C for 2 minutes followed by 35 cycles at 94°C for 45 seconds, 47 °C for 45 seconds and 68°C for 1.3 minutes, and a final extension of 68°C for 10 minutes. The PCR was carried out with

GeneAmp PCR System 9700 (Applied Biosystem, Foster City, CA). Ten microliters of

PCR product were mixed with EZ-Vision three loading buffer (Amresco LLC, Solon,

OH), loaded onto a 1.3% agarose gel and subjected to electrophoresis at 100V for ~ 20 minutes. The results were visualized by exposing the gel to UV light into Fluorchem FC2 multimage II system (Alpha Innotech/Cell Biosciences Inc., Santa Clara, CA).

Phylogenetic Analysis

The 561 bp of PCR-amplified products from selected samples were gel purified with

QIAquick gel extraction kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. The purified amplicons were sequenced using both forward and reverse primers used in PCR. The newly determined sequences together with the previously reported sequences of chicken and turkey parvoviruses available in GenBank, were analyzed with the Megalign program using the Clustal V alignment algorithm

(DNASTAR Lasergene, Madison, WI) and a final phylogenetic tree was constructed using Neighbor-Joining algorithm with a bootstrap test of 1000 replicates.

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2.4 Results

Transmission Electron Microscopy

The TEM was used for the detection of enteric viruses in all samples collected. The TEM analysis of the samples resulted in the identification of single or mixtures of different viruses. The viruses detected were: corona-like viruses, rotaviruses, reoviruses, adenoviruses and small round viruses (SRV) which included viruses ranging from 18 to

30 nm in diameter such as parvoviruses and astroviruses (Table 2.2; Fig. 2.1). Overall,

67.5% of samples contained at least one type of enteric virus and 42% of the samples showed the presence of two or more types of virus particles. The most commonly identified viruses were SRV which were detected in 52% of the turkey samples.

Conventional PCR for the detection of turkey parvoviruses

The same samples used for TEM were tested for parvovirus using NS-gene specific PCR.

The PCR was positive in 70.4%, 69.4%, 100%, 81%, and 66.7% of samples, collected in

2000, 2005, 2008, 2009, and 2010, respectively (Table 2.2). We found that 69.3% of samples that were SRV- positive by TEM were also parvovirus-positive by PCR. On the other hand, 74% of the samples that were SRV-negative by TEM were parvovirus positive by PCR (Table 2.3).

Samples were also analyzed based on two age-groups: poults between 1 and 7 weeks of age and between 8 and 19-week-old. We found a higher prevalence (80%) in the latter age group compare to the former one which had 60% prevalence. 47

Phylogenetic Analysis

The partial NS gene of selected turkey samples (n=15) were sequenced and analyzed with the available sequences of chicken and turkey parvoviruses present in GenBank, and phylogenetic tree was constructed (Fig. 2.2). Partial NS gene sequences (from position

1903 to 2284 in the ChABU-P1 strain (GU214704), 382 bp) were used for comparison and phylogenetic tree construction. The tree indicates a clear dichotomy: one branch clusters the majority of the chicken and few turkey strains together with one of our turkey samples (Chicken group); the other branch clusters the rest of our turkey samples together with three Hungarian samples, one (Ch1515/07/HUN) isolated from chickens and two (Tu/33/2010/HUN and Tu762/2009/HUN) from turkeys (Turkey group). The turkey group share unique amino acids compared to the chicken groups in three positions

(based on the amino acid sequence of ChABU-P1 strain): 574 (Ala vs. Thr); 597 (Thr/Ala vs. Pro); 611 (Gln vs. Glu).

The percentage of nucleotide identity among the our turkey strains, within the turkey group, was between 96.9% and 100%, with the exception of the strain Tu1/PA/10 that shared a lower nucleotide identity (95.5% and 98.7%). In addition, the nucleotide identity of our turkey strains compared to the Hungarian strains, Tu33/2010/HUN and

Tu762/2009/HUN, was between 96.9% and 99.7%; and lower when comparing the strain

Tu1/PA/10, between 95.5 % and 97.1%. Moreover, the chicken strain Ch1515/07/HUN had between 92.7 and 95.8% nucleotide identity with the other strains within the turkey group. On the other hand, the nucleotide identity among the strains within the chicken

48 group was between 89.4 and 95.6 %. One of our turkey strains (Tk4/VA/00) that clustered together with the chicken samples had 87% nucleotide identity with the other turkey stains collected in 2000; and, between 84.6 % and 87.8% with the strains clustered in the turkey group.

2.5 Discussion

Parvoviruses have been identified in chickens and turkeys in Europe and the USA since the early 1980’s. They have been identified in cases of poultry affected by enteritis and stunting, and recently in chicken with cerebellar hypoplasia and hydrocephalus (Kisary,

1984; Marusak et al., 2010; Palade, 2011b; Trampel et al., 1983; Zsak et al., 2009); and also in apparently healthy flocks (Palade, 2011b). The data on parvovirus’ distribution in the US turkey flocks is limited; there is a recent report (Zsak et al., 2009) in which Zsak et al. tested turkey samples collected from 5 states over a 5 year period and chicken samples collected in 7 states over a 3 year period. They found that overall 77% of the chicken and 78% of the turkey samples tested positive for parvovirus (8).

In our study, we examined turkey samples from two additional US states (PA and OH) not included in the previous survey. Moreover, we included samples collected earlier (in

2000) and also recently in 2009-2010 from the US turkey farms for which there is no available information on parvovirus distribution in the literature. Furthermore we tested samples from birds of a wide age range up to 19 weeks. A previous study (Zsak et al.,

2009) analyzed only samples from poults up to 7 weeks of age. Our results indicated a total parvovirus’ prevalence of about 71.6% in the turkey flocks tested. However, when 49 we analyzed the samples taking into consideration the age of the poults, we found a higher prevalence (80%) in poults between 8 and 19 weeks of age compared to the ones between 1 and 7 weeks. The reason for splitting the samples into those age groups is to be able to compare our results with the ones published by other researchers. We do not have both age groups represented in all states and years. The samples from North Carolina are the only ones that have a similar number of samples between the two age groups, while the samples from Virginia are mainly from one age group. Therefore, it is speculated that parvovirus is widespread and its persistence in the turkey population may be longer compared to other enteric viruses. However, additional surveillance studies in both healthy and diseased flocks of varying age from additional states are needed to confirm it.

Moreover, even though we observed 20% lower prevalence in poults less than 7 weeks of age compared to previous study, nevertheless our overall detection rates from different states from varying age of turkeys correlate well with the previous findings. Minor discrepancy may be due to random surveillance conducted in different years and different states.

The phylogenetic analysis showed that overall our turkey samples had a certain degree of variability which is a possible indication of the circulation of different strains. The percentage of nucleotide identity among our turkey strains is, to some extent, lower than the one among the US strains previously detected (Zsak et al., 2009). Unfortunately, those sequences are not available in GenBank for comparison, thus any speculation regarding variability of circulating strains is not possible. Of interest is that two of our turkey strains (Tu38/NC/05 and Tu40/NC/05) collected in the same farm but in different

50 houses share 100% nucleotide identity between each other and with two other strains of

2005 (Tu117/NC/05 and Tu54/NC/05) and one strain of 2000 (Tu1/VA/00). These data suggest the circulation of a similar strain in different regions and years; nonetheless, the comparison of their full genomic sequences is needed to better understand their origin and relationship. On the other hand, even though strains Tu3/OH/09 and Tu4/OH/09, came from different houses within the same farm, they share a lower nucleotide identity

(98.4%) which might indicate the presence of multiple strains in the region. Similar results were found in Hungary where the presence of multiple circulating parvovirus strains within the same farm have been suggested (Palade, 2011b). Overall, the sequence variability within years was similar, with the exception of the year 2000. The three strains sequenced showed low nucleotide identity among themselves; in particular, one strain

(Tu4/VA/00) clustered together with two chickens and one turkey European strain within the chicken group. Previous studies, indicated that chicken and turkey parvoviruses share high amino acid sequence identities, based on the analysis of the full length genome of one chicken and two turkey parvoviruses, which suggested a common ancestor (Day and

Zsak, 2010). Moreover, the phylogenetic analysis of a conserved region of the NS gene showed that they cluster in separate branches, which is a likely indication of a species- specific adaptation (Palade, 2011b; Zsak et al., 2009). The presence of turkey parvoviruses with higher identities with chicken rather than turkey strains could be explained as an inter-species transmission between chicken and turkey. Nonetheless, the comparison of the full length genome of these strains and an experimental transmission study is needed in order to verify this hypothesis.

51

In our study, we used the TEM to test all samples for enteric viruses. TEM has been widely used as a diagnostic tool before the availability of molecular techniques such as

PCR (Doane, 1980) and is still considered an important diagnostic method (Curry, 2006;

Gentile, 2005). In a recent survey of Poults Enteritis in turkeys, Woolcock and

Shivaprasad (Woolcock, 2008) used it as a diagnostic tool for the identification of enteric viruses in more than 2400 turkey samples collected in California during 10 years span.

Moreover, it has been used together with immunohistochemistry and PCR in a study aimed at the detection of chicken and turkey parvoviruses in Hungary (Palade, 2011b).

TEM has various advantages compared to the PCR, such as: detecting the presence of multiple viruses in the same sample; detecting novel viruses for which there are no other tools available for diagnosis and overcome the drawback of highly variable viruses that can escape the detection by PCR due to mutations or recombination. However, it also has disadvantages such as: low detection limits, between 105-8 particles/ ml; and the need for specialized personnel to run the test (Curry, 2006; Gentile, 2005).

In case of SRV, it is not always possible to determine the genus to which these viruses belong due to multiple viruses having similar sizes and structures and the internal structures of the capsid are not always evident. Moreover, PCR is a more sensitive test that can detect low viral load and that would give a false negative result by TEM due to a lower testing sensitivity. As expected, the PCR detected high number of parvovirus positive samples compared to SRV-positive detected by TEM. It is also interesting to note that parvovirus detection rates by PCR were similar between SRV positive and

52 negative samples (Table 3). These results explain the different SRVs involved in enteric disease in turkeys in addition to the lower sensitivity of the TEM in detecting viruses.

In our study, TEM detected multiple viruses in the same sample in more than 60% of fecal samples collected from birds with the history of stunting syndrome, or enteritis, with increase mortality rate. Out of these samples, parvovirus was positive in more than

92% of the cases. These data suggest possible involvement of parvoviruses in the enteric diseases of turkeys; however, their specific role still needs to be defined clearly.

In summary, our results confirm the wide distribution of Parvoviruses in commercial turkey farms. Furthermore, the phylogenetic analysis of partial NS gene sequences showed a certain degree of variability among our turkey samples and their separate clustering from chicken strains with an exception. Lastly, TEM was a valuable method that allowed us to identify mixed infections and should be considered as a useful tool that complements PCR.

2.7 Acknowledgements

We would like to thank the Molecular and Cellular Imaging Center, OARDC/OSU, and in particular Dr. Meulia T., Kaszas A. and Whittier J.

Salaries and research support was provided by state and federal funds appropriated to the

Ohio Agricultural Research and Development Center, The Ohio State University.

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2.6 References

Berns, K.a.P., CR, 2007, Parvoviridae, Vol 2, Fifth Edition. Lippincott Williams and Wilkins, Philadelphia. Curry, A., Appleton, H., Dowsett, B., 2006, Application of transmission electron microscopy to the clinical study of viral and bacterial infections: present and future. Micron 37, 91-106. Day, J.M., Zsak, L., Determination and analysis of the full-length chicken parvovirus genome. Virology 399, 59-64. Doane, F.W., 1980, Virus morphology as an aid for rapid diagnosis. The Yale journal of biology and medicine 53, 19-25. Gentile, M.a.G., H. R., 2005, Rapid viral diagnosis: role of electron microscopy. The new microbiologica, 1-12. Kisary, J., 1985, Experimental infection of chicken embryos and day-old chickens with parvovirus of chicken origin. Avian Pathol 14, 1-7. Kisary, J., Avalosse, B., Miller-Faures, A., Rommelaere, J., 1985, The genome structure of a new chicken virus identifies it as a parvovirus. J Gen Virol 66 ( Pt 10), 2259- 2263. Kisary, J., Nagy, B, Bitay, Z., 1984, Presence of parvoviruses in the intestine of chickens showing stunting syndrome. Avian Pathol 13, 339-343. Marusak, R.A., Guy, J.S., Abdul-Aziz, T.A., West, M.A., Fletcher, O.J., Day, J.M., Zsak, L., Barnes, H.J., 2010, Parvovirus-associated cerebellar hypoplasia and hydrocephalus in day old broiler chickens. Avian Dis 54, 156-160. Palade, E.A., Kisary, J., Benyeda, Z., Mandoki, M, Balka, G., Jakab, C., Vegh, B., Demeter, Z., Rusvai, M., 2011, Naturally occurring parvoviral infection in Hungarian broiler flocks. Avian Pathol 40, 191-197. Trampel, D.W., Kinden, D.A., Solorzano, R.F., Stogsdill, P.L., 1983, Parvovirus-like enteropathy in Missouri turkeys. Avian Dis 27, 49-54. Woolcock, P.R.a.S., H. L., 2008, Electron Microscopic Identification of Viruses Associated with Poult Enteritis in Turkeys Grown in California 1993-2003. Avian Dis 52, 209-213. Zsak, L., Strother, K.O., Day, J.M., 2009, Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses. Avian Dis 53, 83-88. Zsak, L., Strother, K.O., Kisary, J., 2008, Partial genome sequence analysis of parvoviruses associated with enteric disease in poultry. Avian Pathol 37, 435-441.

54

Table 2.1. Information of turkey samples examined

Year of samples Origin (# farms) Age of Birds Health Status of the collection (# of samples) flocks (# samples) 8-day-old (2), 21-day-old (1), 55-day-old (2), 62-day-old (2), 64-day-old (2), 66-day-old (5), 2000 (44) Virginia (14) 72-day-old (2), 76-day-old (2), Unknown 79-day-old (2);80-day-old (4), 85-day-old (2), 94-day-old (4) 95-day-old (4), 121-day-old(2) Unknown age (8)

2005 (98) North Carolina (14) 3-week-old (45) Unknown 19-week-old (53) 41-day-old (1) 2008 (3) Pennsylvania (3) 49-day-old (2) Stunting, high mortality and enteritis 2-week-old (5), 3-week-old (4), Ohio (3) 4-week-old (3), 5-week-old (4) Stunting, increased 2009 (21) Pennsylvania (5) 22-day-old (1), 26-day-old (1), mortality and enteritis 31-day-old (1), 32-day-old (1), 34-day-old (1)

2010 (3) Pennsylvania (3) 28-day-old (2), 42-day-old (1) Stunting, increased mortality and enteritis Total (169)

55

Table 2.2. TEM and PCR Results.

TEM Results Year of PCR Parvovirus samples Parvovirus* PCR

collection positive in Mixed (# samples) mixed Infections infections*** SRV* Rotavirus* Reovirus* Corona-like Adenovirus* out of total Virus* positive samples**

2000 15/44 None None 3/44 None 2/17 31/44 1/2

2005 55/98 33/98 None 27/98 None 32/74 68/98 23/32

2008 3/3 None None 2/3 1/3 3/3 3/3 3/3

2009 15/21 5/21 10/21 None 3/21 10/19 17/21 10/10

2010 0/3 1/3 1/3 None None 1/1 2/3 0/1

Total 88/169 39/169 11/169 32/169 4/169 48/114 121/169 37/48

1 SRV (=small round viruses); * Number of positive samples/total samples examined; ** Number of mixed infections/total enteric viruses positive samples detected by TEM; *** Number of parvovirus positive samples/ total mixed infections detected by TEM.

56

Table 2.3 Correlation between TEM detection of SRV and PCR detection of parvoviruses

PCR + (%) PCR – (%) Total (%)

SRV + (%) 61/88 (69.3 %) * 27/88 (30.7 %)# 88/169 (52%)§

SRV – (%) 60/81 (74 %)** 21/81 (25.9 %)## 81/169

(47.9%)§§

Total (%) 121/169 (71.6%)± 48/169(28.4%)± ±

* Number of parvovirus-positive samples by PCR/Total SRV-positive samples by TEM ** Number of parvovirus-positive samples by PCR/Total SRV-negative samples by TEM # Number of parvovirus-negative samples by PCR/Total SRV-positive samples by TEM ## Number of parvovirus-negative samples by PCR/Total SRV-negative samples by TEM ± Number of parvovirus-positive samples by PCR/Total samples examined ± ±Number of parvovirus-negative samples by PCR/Total samples examined § Number of SRV-positive samples by TEM/Total samples examined §§ Number of SRV-negative samples by TEM/Total samples examined

57

Figure 2.1 Electromicrographs of SRV of 18-20 nm in diameter (A), SRV of 27 nm in diameter (B), Reoviruses (C), Rotaviruses (D), and Adenovirus (E). Magnification 60 kx.

58

Figure 2.2 Cladogram using Neighbor-Joining algorithm and representing the genetic relationship of turkey parvovirus field samples (bolded in the tree) and previously identified strains of chicken and turkey parvoviruses from Europe and USA. For two samples, Tk1/PA/08 and TK54/NC/05, a shorter sequence was used for comparison, 277 nt and 364 nt in length, respectively.

59

60

Chapter 3: Detection of parvoviruses and other enteric viruses in

commercial turkey flocks in four states of the United States between

2000 and 2010.

3.1 Summary

In 2009 Zsak et al. developed a PCR test for the detection of chicken and turkey parvoviruses and found that parvovirus was present in 78% of turkey samples collected in various years in the U.S. (Zsak et al., 2009). In our previous prevalence study (Murgia et al. submitted for publication) we tested 168 samples for parvoviruses by PCR and we found an overall parvovirus prevalence of 71.6 %; moreover, we tested the same samples by transmission electron microscopy and we detected multiple enteric viruses, singly or in combination, with the highest prevalence (52%) of small round viruses. The purposes of the present study are: to determine the prevalence of other enteric viruses in the previously tested samples using PCR and RT-PCR methods, to further analyze the previously collected data on parvovirus using statistical methods and to determine the relationship between parvoviruses and other enteric viruses detected. The 168 samples were tested by PCR for adenovirus and RT-PCR for astrovirus, reovirus, rotavirus, and coronavirus. We found that reovirus was detected with similar prevalence independent of

61 state and year of collection. On the other hand, the prevalence of astrovirus and rotavirus was significantly lower in 2000 and 2005, respectively. A significantly lower rate of co- infection was detected in 2005 versus the other years. The analysis of the samples based on two age groups (1 to 7 weeks and 8 to 19 weeks) showed a significantly higher prevalence of astrovirus, reovirus, rotavirus in samples collected from birds of 1 to 7 weeks of age; on the other hand, although parvovirus was detected with higher prevalence in both age groups, a significant increase in prevalence was identified in samples collected from birds of 8 to 19 weeks of age. Our results confirm the wide distribution of parvovirus in the U.S. Moreover, we found that it is present in turkeys of wide age ranges and this is in contrast with the higher prevalence of the other enteric viruses in young birds. Our data showed a significantly higher prevalence of parvovirus in older birds; however more systematic surveillance studies including larger number of samples from different states and years from flocks of known health status are needed to assess the association of parvovirus and co-infection with other enteric viruses in enteric disease problems in turkeys.

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3.2 Introduction

Parvovirus was identified in cases of enteritis and high mortality in turkeys for the first time in 1983 (Trampel et al., 1983). Since then there were no further reports of parvovirus in turkeys until a PCR test detected parvovirus in 78% of turkey samples collected in various years in the U.S. (Zsak et al., 2009). In that study the health status of the flocks examined was unknown. In our previous prevalence study (Murgia et al. submitted for publication) we tested 168 samples for parvoviruses by PCR and we found an overall parvovirus prevalence of 71.6 %; moreover, we tested the same samples by transmission electron microscopy and we detected multiple enteric viruses, singly or in combination, with the highest prevalence (52%) of small round viruses. Currently, enteric viruses are detected using real-time or conventional PCR and RT-PCR tests as monoplex or multiplex (Culver, 2010; Day et al., 2007; Koci et al., 2000; Pantin-

Jackwood, 2008; Pantin-Jackwood et al., 2007; Sellers et al., 2004; Spackman et al.,

2005; Y. Tang, 2005). Multiplex assays have the advantages of being able to detect multiple targets in the same reaction saving time and money; however, they have to be very carefully optimized in terms of primer design and amplicons length, in order to avoid primers dimers, cross-hybridization of the different primers pairs, aspecific binding and competition among the target templates. The disadvantage compared to the monoplex reactions is that there is a competition among target DNA or RNA being amplified, so if one target is present in lower amount it may not be amplified giving false negative results. The same considerations are valid in case of a monoplex or multiplex real-time; nonetheless the real-time has the following advantages compared to the conventional 63

PCR: it is possible to quantify the reaction product; and there is no need for processing post-reaction. Enteric viruses are involved in the economical important syndromes of poultry known as poultry enteritis complex (PEC) and poultry enteritis and mortality syndrome (PEMS). Therefore, the development of more sensitive and specific diagnostic tests is important to improve their detection and thus gather information on their prevalence in order to develop prevention and control strategies.

The purposes of the present study are: to determine the prevalence of other enteric viruses in the previously tested samples using conventional polymerase chain reaction (PCR) and reverse-transcription- polymerase chain reaction (RT-PCR) methods, to further analyze the previously collected data on parvovirus using statistical methods and to determine the relationship between parvoviruses and other enteric viruses detected.

3.3 Material and Methods

Samples

Samples of feces, litter or intestinal contents, were collected from 2000 to 2010 from

Virginia, North Carolina, Ohio, and Pennsylvania. The age of the birds ranged from 1 to

19 weeks. The samples collected from 2008 to 2010 are from flocks affected by stunting syndrome, high mortality and enteritis; we do not have any information regarding the health status of the flocks for the samples collected in 2000 and 2005. The samples were diluted 1:10 (v/v) in 0.01M Phosphate Buffer Saline (PBS) pH 7.4, clarified at low speed

64 centrifugation and filtered through 0.45 µm syringe filter. The filtered samples were then used for DNA and RNA extraction.

Nucleic acids extraction

Fifty microliters of the filtered samples were subjected to nucleic acid extraction in

MagMAXTM Express Magnetic Particles Processor (Applied Biosystems, Foster City,

CA) by using MagMAXTM-96 Viral RNA Isolation Kit (Ambion/AB, Austin, TX) following the manufacturer’s instructions.

PCR for the detection of adenoviruses

The PCR was performed using published primers, HexF1 and HeXR1(Mase M, 2009) and a PCR Master Mix (Promega, Madison, WI). The primers target is a conserved region in the hexon gene of all three adenovirus groups and gave an 800 bp product. The reaction volume was 25 µl with 0.8 µM of each primer. The PCR cycle was: denaturation at 95°C for 2 minutes followed by 35 cycles at 94°C for 30 seconds, 50 °C for 45 seconds and 72°C for 1.3 minutes, and a final step of 72°C for 10 minutes. The PCR was carried out with GeneAmp PCR System 9700 (Applied Biosystem, Foster City, CA). Ten microliters of PCR product were mixed with EZ-Vision three loading buffer (Amresco

LLC, Solon, OH), loaded onto a 1.3% agarose gel and subjected to electrophoresis at

100V for ~ 20 minutes. The results were visualized by exposing the gel to UV light into

Fluorchem FC2 multimage II system (Alpha Innotech/Cell Biosciences Inc., Santa Clara,

CA).

65

RT-PCR for the detection of astrovirus

The RT-PCR for astrovirus was performed using the published primers TAPG-L1 and

TAPG-R1 which amplify a 601 bp fragment of the polymerase gene (Y. Tang, 2005) and using Promega enzymes and buffers (Promega, Madison, WI). The PCR was carried out with GeneAmp PCR System 9700 (Applied Biosystem, Foster City, CA). The RT-PCR cycle was: RT step for 45°C for 45 minutes followed by denaturation at 95°C for 5 minutes, then 35 cycles at 94°C for 1 minute, 47°C for 45 seconds and 72°C for 1.3 minutes, and a final step of 72°C for 10 minutes. The RT-PCR product post reaction was analyzed as above.

RT-PCR for the detection of reovirus

The RT-PCR for reovirus was performed using the published primers S4-F13 and S4-

R1133, which amplify a 1120 bp fragment of the S4 gene (Pantin-Jackwood et al., 2008) and using Qiagen one step RT-PCR kit (Qiagen, Inc., Valencia, CA.), following the manufacturer’s instructions. The RT-PCR was carried out with GeneAmp PCR System

9700 (Applied Biosystem, Foster City, CA). The RT-PCR cycle was: RT step for 50°C for 45 minutes followed by 94°C for 15 minutes, then 35 cycles at 94°C for 30 seconds,

53°C for 1 minute and 72°C for 1.3 minutes, and a final step of 72°C for 10 minutes. The

RT-PCR product post reaction was analyzed as above.

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RT-PCR for the detection of rotavirus

The RT-PCR for rotavirus was carried out using the published primers NSP4-F30 and

NSP4-R660, amplifying a 630 bp fragment of the NS4 gene (Day et al., 2007), and using

Qiagen one step RT-PCR kit (Qiagen, Inc., Valencia, CA.), following the manufacturer’s instructions. The RT-PCR was carried out with GeneAmp PCR System 9700 (Applied

Biosystem, Foster City, CA). The RT-PCR cycle was: RT step for 50°C for 30 minutes followed by 94°C for 15 minutes, then 35 cycles at 94°C for 45 seconds, 51°C for 1 minute and 72°C for 1.3 minutes, and a final step of 72°C for 10 minutes. The RT-PCR product post reaction was analyzed as above.

RT-PCR for the detection of coronavirus

The RT-PCR for coronavirus was performed using published primers, UTR41+ and

UTR11-, targeting the UTR region of the coronavirus genome (Culver, 2010), and using

Qiagen one step RT-PCR kit (Qiagen, Inc., Valencia, CA.), following the manufacturer’s instructions. The RT-PCR was carried out with GeneAmp PCR System 9700 (Applied

Biosystem, Foster City, CA). The RT-PCR cycle was: RT step for 42°C for 60 minutes followed by 95°C for 15 minutes, then 35 cycles at 94°C for 1 minute, 46°C for 1 minute and 72°C for 2 minutes, and a final step of 72°C for 10 minutes. The RT-PCR product post reaction was analyzed as above.

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Statistical analysis

The comparisons of the prevalence of each virus among years, states, ages, health status, were analyzed using Chi Square Test and Fisher Exact Test using GraphPad Prism 5

Software (GraphPad Software, Inc., La Jolla, CA). We considered a p≤ 0.05 as statistically significant. Logistic regression analysis was used to assess the association between parvovirus and the other enteric viruses using STATA (StataCorp LP, College

Station, TX).

3.4 Results

PCR for the detection of adenovirus

Overall adenovirus was detected in 5% of the tested samples (Fig.3.1). It was detected in samples from 2000 and 2008 to 2010, but not in the samples collected in 2005 in North

Carolina (Fig 3.2 and Fig.3.3). When the samples were analyzed based on the age of the birds we found a higher prevalence in the samples collected from turkeys of 1 to 7 weeks of age (Fig.3.4).

RT-PCR for the detection of astrovirus

The overall prevalence of astrovirus was 46 % (Fig.3.1). There was a significant increase in the detection of astrovirus from 2000 to 2010 (Fig.3.2). The prevalence of astrovirus in

Virginia was significantly lower compared to the other states tested (Fig. 3.3). Moreover,

68 there was a significant difference in prevalence between the two age groups with a higher prevalence in the samples collected from young birds (Fig.3.4).

RT-PCR for the detection of reovirus

Overall reovirus was detected in 37% of the tested samples (Fig.3.1). Moreover, it was detected with similar prevalence independent of state and year of collection (Fig.3.2 and

Fig.3.3), but with higher prevalence in samples collected from birds of 1 to 7 weeks of age (Fig.3.4).

RT-PCR for the detection of rotavirus

Rotavirus had an overall prevalence of 45% (Fig.3.1). However, its prevalence in 2005 in samples collected in North Carolina was significantly lower compared to the other years and states (Fig. 3.2 and Fig.3.3). As for the previous viruses, a statistically significant prevalence was found for samples collected from young birds (Fig.3.4).

RT-PCR for the detection of coronavirus

Coronavirus was detected in 3% of the samples tested (Fig.3.1). There was no statistical significance in the prevalence of coronavirus between the age groups, or among years and states (Fig.3.2 to Fig 3.4).

69

Co-infections of more than one virus

The samples tested showed a high level of co-infections where more than one virus was detected in the same sample. The co-infection rate was statistically significantly lower for the samples collected in 2005 in North Carolina compared to the other years and states

(Fig.3.2 and Fig.3.3). Moreover, the lower rate of co-infections in older birds was statistically significant (Fig.3.4).

PCR for the detection of parvovirus

In the graphs (Fig.3.1 to Fig.3.5), we included the parvovirus PCR data previously obtained (Murgia et al. submitted for publication) for comparison and further analysis.

We found that while there was no significant difference in the prevalence of parvovirus among years and states and it was detected with higher prevalence in both age groups, a significant increase in prevalence was identified in samples collected from birds of 8 to

19 weeks of age.

Correlation between enteric viruses detected by TEM vs. PCR/RT-PCR methods

In our previous study we detected enteric viruses in the samples using TEM, however considering that this method has a low detection limit of 105-6 particle/ml (Curry, 2006;

Gentile, 2005), in the present study, we decided to use the more sensitive and specific molecular techniques of PCR and RT-PCR for the detection of the enteric viruses within the same samples (Table 3.1) (Pantin-Jackwood, 2008; Pantin-Jackwood et al., 2007).

The RT-PCR for astrovirus was more specific compared to the TEM; in fact 41% of

70 samples that were TEM negatives for small round viruses (SRV) were positive by RT-

PCR. On the other hand, it failed to detect astroviruses in 50% of SRV TEM-positive samples; however, this is not surprising considering that the acronym SRV includes, apart from astrovirus, other small viruses with similar diameter and with a not clearly distinctive morphological features. The RT-PCR for reovirus was more sensitive compared to TEM. In fact, 7/8 TEM positives samples were also positive by RT-PCR, moreover, 55/160 TEM negatives were positive by RT-PCR. In the same direction goes the PCR for adenovirus that detected all the TEM positive samples and in addition 7/166 samples that were TEM negative. On the other hand, the RT-PCR for rotavirus was able to detect 40% TEM positive samples, and in addition 47% of TEM-negative ones.

However, in 59% of the cases failed to detect TEM positive samples. Unexpectedly the

RT-PCR for coronavirus failed to detect coronavirus in 100% of TEM-positive samples and it could detect only 4% of coronavirus in TEM-negative samples.

Association between parvovirus and other enteric viruses

We used logistic regression analysis to assess the level of association between parvovirus and the other enteric viruses detected in the same sample. No significant association was found when controlling for other variables.

3.5 Discussions

It is important to determine the prevalence of enteric viruses in commercial turkey operations in order to develop specific control and prevention strategies. In a longitudinal

71 study in 2005 Pantin-Jackwood et al. collected samples from 2, 4, 6, 8, 10, 12 week-old healthy turkey poults in eight turkey flocks in North Carolina and one research facility at

North Carolina State University. They found a prevalence of 89.5% and 67.7 % for astrovirus and rotavirus, respectively (Pantin-Jackwood et al., 2007). Moreover, the same group performed another survey of healthy chicken and turkey flocks in various US states between 2005 and 2006 collecting samples from birds of less than two weeks and few samples from turkeys between 2 and 6 weeks of age. They found that, in turkeys, the prevalence of astrovirus, rotavirus and reovirus, was 100% , 69.7 % and 45.5 %, respectively; moreover, these viruses were present singly and in combination (Pantin-

Jackwood et al., 2008). In our study we collected samples from turkeys of a wide age range, from 1 to 19 weeks; moreover, we tested samples from 2000 and 2008 to 2010, for which, in the literature, there are no information on turkey enteric viruses’ prevalence.

We found that the overall prevalence of astrovirus and rotavirus was similar, and it was lower than the one reported in the above mentioned studies. However, if we look at the distribution of the viruses in the two age groups we found that in turkeys of 1 to 7 weeks of age the prevalence of astrovirus and rotavirus was 84% and 54%, respectively, which is similar to the one previously reported for birds of the same age group. In addition, this difference in the detection of astrovirus between young and old birds is in agreement with the fact that poults less than three weeks of age are the most susceptible to its infection

(Reynolds, 2003). Nevertheless, it is of note that in the previous survey (Pantin-Jackwood et al., 2007), astrovirus were detected up to 12 weeks in almost all the farms tested, which is in disagreement with our finding of a significant drop in astrovirus detection in

72 older birds, from 84% to 13%. Astrovirus is highly resistant in the environment, therefore it is possible that once it enters in the farm it persists for long time consequently birds can be continuously re-infected and this can be detected in a longitudinal study. In our study, we collected the samples at only one time point and it is possible that the flock did not contain astrovirus in the first place; in addition, samples of different age were collected in different farms. Reovirus, adenovirus and coronavirus’ prevalence detected in our study are in agreement with the ones in previous surveys. Of note is that the prevalence of parvovirus is not affected by the age of the birds as previously reported (Murgia et al., submitted for publication); moreover, it is significantly higher in older poults, which is in contrast with the other enteric viruses usually affecting young birds within three weeks of age. This data suggests that parvovirus might persists for long time in the farm; however, more systematic surveillance studies including larger number of samples from different states and years are needed to confirm it.

The overall co-infection rate was significantly lower in older birds compared to younger birds. Unfortunately the health status of the turkeys of 8-19 weeks of age was unknown; therefore any potential association with disease is not possible. On the other hand, 93% of the samples collected from birds experiencing enteric diseases showed co-infections with two or more viruses (Fig.3.5), suggesting a possible synergistic effect among enteric viruses in the severity of the disease’s outcome.

The use of molecular techniques versus the classical TEM has the advantage of high sensitivity, however, it can give false negative results in case of virus that are prone to mutations, such as RNA viruses. In this study we re-tested, for enteric viruses, samples

73 previously tested by TEM for two reasons: first to determine the genus of the small round viruses previously identified by TEM and second to be able to detect the viruses present in the sample at a concentration below the TEM detection limit. The RT-PCR was a more sensitive method, compare to TEM, for the detection of reovirus and astrovirus, and the

PCR for the detection of parvovirus and adenovirus. However, RT-PCR methods failed to detect rotaviruses in 60% of the TEM positive samples; this can be due to variation in the target sequence of NSP4 gene in the circulating strains. This it is not surprising because rotaviruses are known to be prone to point mutations, recombination and reassortment, that can influence the sensitivity of the molecular tests. More remarkable case was the one for coronavirus when the RT-PCR was unable to detect all TEM positive samples.

Circulation of different strains and mutations can explain these data (Culver, 2010).

In conclusion, we showed an age dependent distribution of the different enteric viruses toward young birds, with the exception of parvovirus which is widely distributed in both age groups, but had a significantly higher prevalence in older birds. In samples collected from diseased birds the rate of co-infections was high suggesting an involvement of multiple viruses in the pathogenesis of enteric diseases. However, more systematic studies with higher number of samples, different states and years are needed to confirm the synergistic effect of the enteric viruses in turkey affected by enteric diseases.

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3.6 Acknowledgements

We would like to acknowledge Dr. LeJeune and Dr. Rodriguez-Palaci for the help with the statistics. Salaries and research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State

University.

3.7 References

Culver, F. A., Britton, P., and Cavanagh, D. (2010). RT-PCR Detection of Avian Coronaviruses of Galliform Birds (Chicken, Turkey, Pheasant) and in a Parrot, Humana Press, New York, NY. Day, J. M., E. Spackman, et al. (2007). "A multiplex RT-PCR test for the differential identification of turkey astrovirus type 1, turkey astrovirus type 2, chicken astrovirus, avian nephritis virus, and avian rotavirus." Avian Dis 51(3): 681-684. Mase M, M. H., Inoue T, Imada T. (2009). "Identification of group I-III avian adenovirus by PCR coupled with direct sequencing of the hexon gene. ." J Vet Med Sci. 71(9): 1239-1242. Pantin-Jackwood, M. J., J. M. Day, et al. (2008). "Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006." Avian Dis 52(2): 235-244. Pantin-Jackwood, M. J., E. Spackman, et al. (2007). "Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms." Avian Dis 51(3): 674-680. Reynolds, D. L. a. S.-C., S.L. (2003). Astrovirus infections, Iowa State University Press. Trampel, D. W., D. A. Kinden, et al. (1983). "Parvovirus-like enteropathy in Missouri turkeys." Avian Dis 27(1): 49-54. Y. Tang, M. M. I., and Y. M. Saif (2005). "Development of Antigen-Capture Enzyme- Linked Immunosorbent Assay and RT-PCR for Detection of Turkey Astroviruses." AVIAN DISEASES 49: 182-188. Zsak, L., K. O. Strother, et al. (2009). "Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses." Avian Dis 53(1): 83- 88.

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Table 3.1 Correlation between transmission electron microscopy (TEM) and PCR/RT-

PCR for the detection of enteric viruses in the tested samples. A. TEM vs. RT-PCR for

astrovirus. B. TEM vs. RT-PCR for reovirus. C. TEM vs. RT-PCR for rotavirus.

D. TEM vs. RT-PCR for coronavirus. E. TEM vs. PCR for adenovirus

A RT-PCR RT-PCR B RT-PCR RT-PCR Astro pos. Astro neg. Reo pos.(%) Reo neg. (%) (%) (%) EM-SRV 44/88* 44/88** EM-Reo pos. 7/8* 1/8 ** (50%) (50%) (87%) pos. (12%) # # # EM-SRV 33/80 47/80 EM- Reo 55/160 # 105/160 # # neg. (41%) (59%) neg. (34%) (66%)

C RT-PCR RT-PCR RT-PCR RT-PCR D Rota pos.(%) Rota neg.(%) Corona pos. Corona neg. (%) (%) EM-Rota 15/37* 22/37** pos. (40.5%) (59.5%) EM-Corona 0 41/41** pos. (100%) EM- Rota # ## 61/131 70/131 EM-Corona 5/127# 122/127## neg. (47%) (53%) neg. (4%) (96%)

PCR Adeno PCR Adeno E pos. neg.

EM-Adeno 2/2* 0 pos. (100%)

EM- Adeno 7/166# 159/166## 76 neg. (4%) (96%)

* Number of parvovirus-positive samples by PCR/Total Virus-positive samples by TEM ** Number of parvovirus-negative samples by PCR/Total Virus-positive samples by TEM # Number of parvovirus-positive samples by PCR/Total Virus-negative samples by TEM ## Number of parvovirus-negative samples by PCR/Total Virus-negative samples by TE

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Prevalence 5% 3% Parvovirus Astrovirus 45% 72% Reovirus Rotavirus 37% Adenovirus 46% Coronavirus

Figure 3.1 Overall prevalence of the tested enteric viruses

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90% 80% 70% Parvovirus 60% Astrovirus Reovirus 50% Rotavirus 40% Adenovirus 30% Coronavirus 20% Co-infections 10% 0% 2000 2005 2008-2010

Figure 3.2 Prevalence of the tested viruses by year

Indicate statistical significance

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100% 90%

80% Parvovirus 70% Astrovirus 60% Reovirus 50% Rotavirus 40% Adenovirus 30% Coronavirus 20% co-infections 10% 0% VA NC PA OH

Figure 3.3 Prevalence of the tested viruses by State

Indicate statistical significance

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90% 80% 70% Parvovirus Astrovirus 60% Reovirus 50% Rotavirus 40% Adenovirus 30% Coronavirus 20% Co-infections 10% 0% 1-7 wks 8-19 wks

Figure 3.4 Prevalence of the tested viruses by Age

Indicate statistical significance

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100% 90% Parvovirus 80% 70% Astrovirus 60% Reovirus 50% Rotavirus 40% Adenovirus 30% Coronavirus 20% Co-infections 10% 0% Unknown Diseased

Figure 3.5 Prevalence of the tested viruses by health status of the flock

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Chapter 4: Development of a Real-Time PCR for the detection of turkey

parvoviruses

4.1 Summary

Chicken and Turkey parvoviruses have been reported in turkeys and in chickens for the first time in the early 1980’s. Their detection was based on their morphologic identification by transmission electron microscope. In 2009, a PCR test, with primers targeting the NS gene of the chicken and turkey parvoviruses, was developed and used in surveys aiming at defining the prevalence of parvoviruses in the US and in Europe.

The aim of our study was to develop a sensitive and specific test for the detection of turkey parvoviruses in fecal samples. Specific primers amplifying a 750 bp product within the NS gene were designed. The PCR product was cloned and the copy number of the plasmid-containing insert was serially diluted 10-fold to obtain a standard curve.

Moreover, negative feces were spiked with 10-fold dilutions of plasmid and the DNA was extracted and used to obtain a standard curve. These dilutions were then used to examine inter- and intra-assay repeatability. Nucleic acids from different viruses and bacteria were not amplified. The real-time was compared with a conventional PCR using the same primer pairs to determine the cutoff of the reaction. The real-time using the plasmid copy number spiked into negative feces showed high variability at the dilution

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101 in the intra and inter-assay repeatability tests , therefore we consider its cutoff to be at the 102 dilution. This cutoff is the same as the conventional PCR. When testing field fecal samples, the two tests were comparable. In conclusion, even though the two tests have the same sensitivity, the real-time PCR had some advantages compared to the conventional PCR which are that it is faster and it is possible to quantify the DNA within the sample.

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4.2 Introduction

Chicken and Turkey parvoviruses belong to the genus parvovirus of the subfamily

Parvovirinae within the family Parvoviridae. The icosahedral parvovirus capsid has a diameter of approximately 18 to 26 nm and contains a 4-6 kb single strand DNA of negative polarity (Berns, 2007). The first reports of parvoviruses in turkeys and in chickens were in 1983 and 1984, respectively (Kisary, 1984; Trampel et al., 1983). Since then there were no further reports of chicken or turkey parvoviruses until 2008 when the use of random PCR allowed, Zsak et al., to determine the partial genome sequence of chicken and turkey parvoviruses (Zsak et al., 2008). The same group then developed a

PCR test with primers targeting the NS gene of the chicken and turkey parvoviruses

(Zsak et al., 2009) and determined the complete genome sequence of one chicken and two turkey parvovirus strains (Day and Zsak, 2010). In 2010 and 2011, there were two further reports of parvoviruses, respectively, a parvovirus-associated cerebellar hypoplasia and hydrocephalus in broiler chickens (Marusak et al., 2010) and a survey reporting the presence of parvovirus in chicken and turkey flocks in Hungary (Palade,

2011b). Moreover, in our laboratory we performed a prevalence study and we found an overall prevalence of parvovirus of 71.6 % in commercial turkey flocks in four US states

(Murgia et al., submitted for publication). The aim of our study was to develop a sensitive and specific test for the detection of turkey parvoviruses in fecal samples.

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4.3 Material and Methods

Samples

Turkey Astrovirus (TastV), Turkey Coronavirus (TCV), and Turkey Adenovirus fecal samples, field fecal samples were from our collection (Table 4.1); E.coli strain and

Cronobacter sakazakii (ATCC No. 29544) DNA were a kind gift from Dr. LeJeune;

Campilobacter coli (ATCC) DNA was a kind gift from Dr. Rajashekara.

DNA extraction

The TastV87, TCV, Turkey Adenovirus and field samples were diluted 1:10 (v/v) in

0.01 M Phosphate Buffer Saline, clarified at low speed centrifugation and filtered through

0.45 μm syringe filter. Fifty microliter of the samples filtered were subjected to nucleic acid extraction in MagMAXTM Express Magnetic Particles Processor (Applied

Biosystems, Foster City, CA) by using MagMAXTM-96 Viral RNA Isolation Kit

(Ambion/AB, Austin, TX) following the manufacturer’s instructions.

Primers design

The set of primers was designed based on the sequence of the non structural protein gene of ABU parvovirus (GenBank Accession No EU304808.1) by using a primers’ design software primer-BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The selected primers, designated P-Fw (5′-ACGAGCACGATGCGTGGGAA-3′) and P-Rv (5′-

CAGGCTGGGCACACGGTCAG-3′), amplifying a 750 bp fragment, were synthesized and HPLC-purified by Integrated DNA Technologies, Inc. (Coralville, IA). 86

Conventional PCR

The PCR was performed using a PCR Master Mix (Promega, Madison, WI) following the manufacturer’s instructions in a 25 ul volume with 1uM of each primer. The PCR was optimized determining the annealing temperature that resulted in a single specific band.

The optimum cycle was established as: denaturation at 95°C for 2 minutes followed by

40 cycles of 95°C for 15 seconds, 69 °C for 33 second and 72°C for 33 seconds. The

PCR was carried out with GeneAmp PCR System 9700 (Applied Biosystem, Foster City,

CA). Ten microliter of PCR product was loaded onto a 1.3% agarose gel and subjected to electrophoresis at 100V for ~ 20 minutes. The band was visualized by exposing the gel to

UV light into Fluorchem FC2 multimage II system (Alpha Innoctech/Cell Biosciences

Inc., Santa Clara, CA)

Real-Time PCR and Standard curve

The 750 bp band was gel purified with Qiaquick Gel Extraction Kit (Qiagen, Valencia,

CA). The purified product was then cloned using Topo XL Cloning Kit (Invitrogen,

Carlsbad, CA). The plasmid containing the 750 bp insert (pl-pv) was purified with

Plasmid purification kit (Qiagen, Valencia, CA), and sequenced using M13-F primer. All the above procedures were conducted following the manufacturer’s instructions. The

Blast search on the sequence matched all chicken and turkey parvoviruses NS protein gene sequences present in GenBank. The DNA concentration of pl-pv was measured in triplicate at the NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific Inc.,

Wilmington, DE) and the mean of the three measurements was then converted into copy

87 number using an online software program

(http://www.uri.edu/research/gsc/resources/cndna.html). A serial 10-fold dilution of the plasmid copy number, in nuclease-free water (Promega, Madison, WI), from 109 to 101 with the dilution 101 further diluted 1:2, 1:4, 1:8, were used to determine the detection limit of the Real-Time vs. the conventional PCR.

To determine the detection limit of the Real-Time PCR in feces, we spiked known copy number of pl-pv, from 109 to 101, into negative feces from SPF turkey poults. The DNA was extracted as described above. Real-Time PCR was performed using GoTaq® qPCR

Master Mix (Promega, Madison, WI), which contains a double strand DNA binding dye.

The reaction mix was: 12.5µl of Master Mix, 0.2 µM of each primer, nuclease free water up to 23 µl and 2 µl of DNA template. The same samples were subjected to the conventional PCR, which was performed as described above. The Real-Time reactions were carried out with a 7500 Real-Time PCR System thermocycler (Applied Biosystem,

Foster City, CA). The following cycle was applied for both Real-Time and conventional

PCR: 95°C for 2 minutes followed by 40 cycles of 95°C for 15 seconds, 69 °C for 33 second and 72°C for 33 seconds. The real-time PCR cycle was followed by a dissociation curve analysis. The detection limit of the Real-Time PCR vs. the conventional PCR was compared.

Sensitivity, Specificity, Intra-assay and Inter-assay Repeatability

We tested the sensitivity of the real-time PCR in comparison with the conventional PCR, using the same primer pairs, by determining the lower detection limit of the two tests. We

88 tested different viruses and bacterial nucleic acids to establish the specificity of the tests.

We used the negative feces spiked with known pl-pv copy number, from 109 to 101, to determine the intra and inter assay repeatability. For the intra-assay repeatability we performed the pl-pv dilutions in triplicate in the same run and we calculated the percentage of the coefficient of variation (Table 4.2). The inter-assay repeatability was carried out by calculating the percentage of the coefficient of variation among three independent runs, each with reactions in duplicate (Table 4.3). Lastly, we calculated the kappa agreement test between the real-time PCR and the conventional PCR.

Statistical analysis

Calculation of the correlation coefficient (r2) for the standard curve was done with the software of the 7500 Real-Time PCR System thermocycler and using the Ct values.

Calculation of the Coefficient of Variation (CV) for the inter- and intra-assays was done using Microsoft Office Excel 2007 (Microsoft Corporation, Redmond, WA), using the formula: (standard deviation/mean) x 100. Calculation of the Kappa agreement test was done by using STATA (StataCorp LP, College Station, Texas).

4.4 Results and discussion

Using the real-time PCR with pure pl-pv plasmid we were able to detect up to 8 DNA copies (Ct= 32.25 ± 0.41), while the conventional PCR detected between 101 and 102 copies. The standard curve was done using the dilution from 106 (Ct= 14.36 ± 0.007) to

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101 (Ct= 29.89 ± 0.33) with a good r2 of 0.991972 (Fig 4.1). When negative SPF turkey feces were spiked with known concentration of pl-pv dilutions, the real-time PCR was able to detect all the dilutions, with the lowest being 101 (Ct= 34 ± 0.33) while the conventional PCR detected only up to 102 DNA copies. Moreover, we used the dilutions from 106 (Ct= 17.09 ±0.05) to 101 (Ct= 34 ± 0.33) to construct a standard curve and also in this case we obtained a good r2 of 0.993875 (Fig 4.2). None of the other viral and bacterial samples were amplified with either real-time or conventional PCR. The dissociation curve showed melting temperature (Tm) ranging from 82.2ºC to 82.8ºC.

The percentage of the CV for the intra-assay and inter-assay repeatability test showed that there was minimal variation along the dilutions except for the dilutions of 101 (Table

4.2 and 4.3). Considering the high variability of the reaction at the dilution 101, we consider the real-time cutoff to be at the dilution 102 (Ct = 34). However, when testing the samples we found out that even the later cycles resulted in specific amplification in the majority of the cases, as confirmed by the identification of a PCR product of the right size when performing agarose gel electrophoresis. Moreover, we observed that the amplification products were present when the melting temperature was between 82◦C and

83.8◦C, while higher or lower Tm resulted in negative results. Due to these considerations, when testing the field samples, we consider the sample positive if the ct value was less or equal to 39 and its Tm was between 82◦C and 83.8◦C. We also confirmed the result by agarose gel electrophoresis. The real-time and the conventional

PCR gave similar results when fecal field samples were tested (Table 4.4). The kappa

90 agreement test showed that the two tests have 90 % agreement beyond chance with a kappa value of 0.7244.

In conclusion, real-time PCR and conventional PCR have the same level of sensitivity, in fact, due to the high variability at the lowest dilution, as demonstrated by the intra and inter-assay repeatability, the detection limit of the real-time reaction was considered to be

102. Even though the two tests have the same sensitivity, the real-time PCR had some advantages compared to the conventional PCR which are that it is faster and it is possible to quantify the DNA within the sample.

4.5 Acknowledgements

We would like to acknowledge Dr. Q. Wang for the suggestions during the optimization steps; Dr. LeJeune and Dr. Rajashakara for providing the bacterial DNA used in the specificity assay; Dr. Meulia, A. Kaszas, and J. Whittier at the Molecular and Cellular

Imaging Center, OARDC/OSU.

Salaries and research support was provided by state and federal funds appropriated to the

Ohio Agricultural Research and Development Center, The Ohio State University.

4.6 References

Berns, K. a. P., CR (2007). Parvoviridae. Philadelphia, Lippincott Williams and Wilkins. Day, J. M. and L. Zsak "Determination and analysis of the full-length chicken parvovirus genome." Virology 399(1): 59-64. Kisary, J., Nagy, B, Bitay, Z. (1984). "Presence of parvoviruses in the intestine of chickens showing stunting syndrome." Avian Pathol 13: 339-343.

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Marusak, R. A., J. S. Guy, et al. (2010). "Parvovirus-associated cerebellar hypoplasia and hydrocephalus in day old broiler chickens." Avian Dis 54(1): 156-160. Palade, E. A., Kisary, J., Benyeda, Z., Mandoki, M, Balka, G., Jakab, C., Vegh, B., Demeter, Z., Rusvai, M. (2011). "Naturally occurring parvoviral infection in Hungarian broiler flocks." Avian Pathol 40(2): 191-197. Trampel, D. W., D. A. Kinden, et al. (1983). "Parvovirus-like enteropathy in Missouri turkeys." Avian Dis 27(1): 49-54. Zsak, L., K. O. Strother, et al. (2009). "Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses." Avian Dis 53(1): 83- 88. Zsak, L., K. O. Strother, et al. (2008). "Partial genome sequence analysis of parvoviruses associated with enteric disease in poultry." Avian Pathol 37(4): 435-441.

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Table 4.1 Information on turkey samples examined

Samples Origin Age of births

2005 (42) North Carolina 3-week-old & 19-week-old

2008 (3) Pennsylvania 49-day-old

2009 (22) Ohio between 2-week-old & 5-week-

Pennsylvania old

2010 (3) Pennsylvania 28-day-old & 47-day-old

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Table 4.2 Intra-assay Repeatability

Copy number of Plasmid- CV % spiked feces 9 10 (12.37 ± 0.11)* 0.92

8 10 (13.65 ± 0.05) 0.37

107 (14.36 ± 0.05) 0.35

106 (17.08 ± 0.14) 0.80

105 (20.52 ± 0.21) 1.04

104 (24.07 ± 0.23) 0.94

103 (27.85 ± 0.23) 0.94

102 (32.26 ± 0.61) 1.9

101 (22.79 ± 19.74) 86.61

* Mean Ct values ± Standard deviation

CV (Coefficient of variation) = (standard deviation/mean) x 100

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Table 4.3 Inter-assay Repeatability

Copy number of plasmid- CV % spiked feces 109 (12.39 ± 0.53)* 0.53

108 (13.66 ± 0.03) 0.27

107 (14.31 ± 0.03) 0.19

106 (17.10 ± 0.04) 0.22

105 (20.39 ± 0.22) 1.09

104 (24.16 ± 0.11) 0.49

103 (27.89 ± 0.15) 0.53

102 (31.81 ± 0.60) 1.8

1 10 (23.13 ± 17.95) 77.60

*Mean Ct values ± Standard deviation

CV (Coefficient of variation) = (standard deviation/mean) x 100

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Table 4.4 Real-Time PCR vs. Conventional PCR detection of fecal samples.

Samples Real-Time PCR Conventional PCR

2010 1/3 * 3/3

2009 16/22 16/22

2008 2/3 3/3

2005 32/42 34/42

Total 51/70 56/70

*Number of positive samples/total samples tested

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Figure 4.1 Standard curve of pure plasmid copy number dilutions

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Figure 4.2 Standard curve of negative feces spiked with copy number of plasmid dilutions

98

Chapter 5: Experimental infections of SPF turkey poults with

parvovirus and astrovirus.

5.1 Summary

Astrovirus and parvovirus have been identified in cases of enteritis as well as in healthy turkey and chicken flocks. Although the pathogenicity of astrovirus for turkeys has been demonstrated in various experimental conditions, the pathogenicity of parvovirus for turkeys has not yet been determined. The aim of our study was to determine the potential pathogenicity of parvovirus in young turkeys. Since we were not able to purify parvovirus from astrovirus due to similar size and density, we compared the pathogenesis of parvovirus and astrovirus co-infection with that of astrovirus infection alone using 2-4- week-old specific pathogen free (SPF) turkeys. In two trials, minimal or no clinical signs and physiological changes in the intestine were observed both in co-infected and astrovirus alone infected birds. However, we observed clear pattern of viral replication of two viruses where astrovirus was detected at early time points after infection (< 14 days post infection (DPI) in trial 1 and <12 DPI in trial 2), while parvovirus in the latter time points (>21 DPI in trial 1 and >12 DPI in trial2). Our study demonstrates potential interference of two enteric viruses in turkeys and additional studies using different age of birds are warranted to further explore the potential impact of parvovirus in turkeys. 99

5.2 Introduction

Parvoviruses and astroviruses are frequently identified in commercial turkey flocks.

Parvoviruses were identified for the first time in 1983 in the ileum of turkeys affected by stunting, enteritis and high mortality (Trampel et al., 1983). In the following year, they were also detected in Hungary in chickens affected by stunting syndrome (Kisary, 1984).

There were no further report of parvovirus in poults until 2009 when a PCR test was developed and used to perform a survey of commercial chicken and turkey flocks in the

U.S. and found a parvovirus prevalence of 77% and 78% in chicken and turkeys flocks, respectively (Zsak et al., 2009). We also performed a prevalence study (Murgia et al. submitted for publication) in which we tested 168 fecal samples collected from 42 turkey farms in four different states in the US between 2000 and 2010 and found an overall parvovirus prevalence of 71.6 %. In Hungary Palade et al. reported the presence of parvovirus in broiler flocks affected by enteric diseases as well as in two healthy flocks

(Palade, 2011b). In addition, parvoviruses were also reported in association with cerebellar hypoplasia and hydrocephalus in broiler chickens (Marusak et al., 2010). On the other hand, astroviruses were identified for the first time in 1980 in UK (McNulty

MS, 1980) and in 1986 in the US (Reynolds and Saif, 1986). Since then they have been routinely detected in surveillance studies of commercial turkey flocks in the US and in

Europe (Domanska-Blicharz et al., 2011; Jindal N, 2010; Pantin-Jackwood, 2008; Pantin-

Jackwood et al., 2007; Reynolds DL, 1987; Saif, 1985).

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Astrovirus and parvovirus are both naked viruses with a capsid’s diameter of 27-30 nm and 18-26 nm, respectively. In previous pathogenesis studies, the infection with astrovirus alone resulted in various degrees of clinical signs and microscopical lesions depending on the strain studied. The clinical signs most commonly found were diarrhea, depression, huddling, agitation and increase vocalization, retarded growth, in birds up to two weeks post infection (Mor et al., 2011; Pantin-Jackwood MJ, 2008; Spackman et al.,

2010; Y. Tang, 2006). The histopathological analysis revealed either no lesions or minimal to mild lesions at early time points. The lesions most commonly found were: lymphocytic depletion, atrophy of the bursa; mild crypt hyperplasia, villus atrophy, and lymphocytic depletion (Pantin-Jackwood MJ, 2008; Spackman et al., 2010; Y. Tang,

2006). Experimental infections where astrovirus was present in conjunction with reovirus or rotavirus or both, resulted in similar clinical signs and lesions as the virus alone

(Spackman et al., 2010). In some cases a significant decrease in weight gain was also observed in group infected with astrovirus and rotavirus (Jindal et al., 2009a, b).

The chicken parvovirus ABU strain was used in two different experimental infections of specific pathogen free (SPF) white leghorn and broiler chickens with different results. In one case, the infection resulted in 40% growth retardation in the broiler chickens, but there was no effect in the SPF white leghorn chicken (Kisary, 1985a). In the other case, no growth retardation nor clinical signs were observed in either SPF white leghorn or broiler chickens (Decaesstecker et al., 1986). The pathogenicity of parvovirus for turkeys has not yet been determined.

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Due to the small difference in diameter and density of astrovirus and parvovirus, our attempts to separate them via serial filtrations or cesium chloride purification were unsuccessful. Therefore, in the present study, we used an inoculum containing both astrovirus and parvovirus to evaluate the potential pathogenicity of parvovirus. We compared the pathogenesis of parvovirus together with astrovirus co-infection with that of astrovirus infection alone using 2-4 week-old turkeys.

5.3 Material and Methods

Viruses

Turkey Astrovirus ’87 (TAstV87) strain was isolated in our laboratory and previously characterized (Tang and Saif, 2004; Y. Tang, 2006). The TAstV87 used in this study was prepared by experimental infection of 2-week-old turkeys and collection of intestinal content at 7 DPI. The intestinal sample Tu/PA/09, containing a mixture of astrovirus and parvovirus, was obtained from Pennsylvania in 2009 from turkeys experiencing enteric disease.

Turkey poults

All SPF turkey poults were from the SPF turkey flock maintained in our facility at the

Ohio Agricultural Research and Development Center/Ohio State University, Wooster,

Ohio. The flock is maintained free from known turkey pathogens including enteric viruses. Before inoculation, the poults were housed in cages with water and feed available ad libitum.

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Experimental infections

Trial 1

Twenty-four 4-week-old SPF turkey poults were randomly assigned to three groups: a non-inoculated group (group 1), a group inoculated with astrovirus (group 2) and a third group inoculated with astrovirus and parvovirus (group 3). Each group was composed of eight poults. The two inoculated groups were housed in separate rooms with high efficiency particulate air-filtered intake and exhaust air. The groups were housed on the floor with wood shavings; water and feed were available ad libitum. The poults of the group 1 were housed in a cage in a room in a separate building with unlimited access to feed and water. The weight of the poults was recorded before inoculation. The inoculated groups were orally inoculated with 0.5 ml of their respective inoculum. Poults were observed daily for clinical signs and two birds per time point per group were euthanized at 7, 14, 21, 28 days post-infection (DPI). At euthanasia, the weight of the poults was recorded and each poult was observed for pathological lesions. The whole gut was removed and a portion of the duodenum, jejunum and ileum were placed in 10% neutral formalin for histopathology; the remaining parts of the gut were saved for nucleic acid extraction and PCR/RT-PCR analysis. Two portions of thymus, bursa, spleen, bone marrow, pancreas, kidney, liver and lung were collected, one to be used for nucleic acid extraction and PCR/RT-PCR analysis; the other was placed in 10% neutral formalin for histopathology.

103

Trial 2

Eighteen 2-week-old SPF turkey poults were randomly assigned to three groups similar to the trial 1. Each group was composed of six poults. The two inoculated groups were housed in the same room as in trial 1; however, the two groups that shared the same room were physically separated from each other. For all groups, feed and water was available ad libitum. The weight of the poults was recorded before inoculation. The inoculated groups were orally inoculated with 0.5 ml of their respective inoculum. Poults were observed daily for clinical signs and two birds per time point per group were euthanized at 7, 12, 18 DPI. At euthanasia, the weight of the poults was recorded and each poult was observed for pathological lesions. The whole gut was removed and a portion of the duodenum, jejunum and ileum were placed in 10% neutral formalin for histopathology; the remaining parts of the gut were saved for nucleic acid extraction and PCR/RT-PCR analysis. Two portions of thymus, bursa, spleen, bone marrow, pancreas, kidney, liver and lung were collected, one to be used for nucleic acid extraction and PCR/RT-PCR analysis; the other was placed in 10% neutral formalin for histopathology.

Nucleic acids extraction

The Intestinal contents of each gut was collected and diluted 1:10 in HBSS, frozen and thawed once, then clarified at 2000 xg using an Allegra X-15R centrifuge (Beckman

Coulter Inc., Fullerton, CA) for 30 minutes at +4°C. The supernatant was collected and stored at – 20 °C until used. The organs were diluted 1:5 in Hank’s balanced salt solution

(HBSS), homogenized with Cole-Parmer LabGen 125 homogenizer (Cole-Parmer,

104

Vernon Hills, IL), frozen and thawed once, then clarified as above. The supernatant was collected and stored at – 20°C until used. Fifty microliters of the sample supernatant were subjected to nucleic acid extraction in MagMAXTM Express Magnetic Particles Processor

(Applied Biosystems, Foster City, CA) by using MagMAXTM-96 Viral RNA Isolation Kit

(Ambion/AB, Austin, TX) following the manufacturer’s instructions.

RT-PCR for Astrovirus

The RNA was subjected to a one step RT-PCR using the published primers TAPG-L1 and TAPG-R1 (Y. Tang, 2005) amplifying a 601 bp fragment of the turkey astrovirus polymerase gene. The reaction volume was 25 µl with 1µM of each primer and was performed using AMV-RT, Go Flexi Taq polymerase and buffers from Promega

(Promega, Madison, WI) and dNPTs mix from Invitrogen (Life Technologies, Grand

Island, NY). The RT-PCR cycle was as follows: 45 minutes at 45°C followed by 5 minutes at 95°C, then 35 cycles of 94°C for 1 minute, 47 °C for 45 seconds and 72°C for

1.3 minutes, and a final step of 72°C for 10 minutes. The RT-PCR reaction was carried out with GeneAmp PCR System 9700 (Applied Biosystem, Foster City, CA).Ten microliters of PCR product were loaded onto a 1.3% agarose gel and subjected to electrophoresis at 100V for ~ 20 minutes. The band was visualized by exposing the gel to

UV light into Fluorchem FC2 multimage II system (Alpha Innoctech/Cell Biosciences

Inc., Santa Clara, CA).

105

Real-Time PCR for parvovirus

The PCR was performed using P-Fw (5′-ACGAGCACGATGCGTGGGAA-3′) and P-Rv

(5′-CAGGCTGGGCACACGGTCAG-3′) primers prepared and validated at our laboratory and a Real-time PCR Master Mix (Promega, Madison, WI). The primers target a conserved region in the NS gene and give a 750 bp product. The reaction volume was

25 µl with 0.2 µM of each primer. The PCR was: denaturation at 95°C for 2 minutes followed by 40 cycles at 95°C for 15 seconds, 69°C for 33 seconds and 72°C for 33 seconds, followed by a dissociation curve. The Real-Time PCR was carried out with the

7500 Real-Time PCR System thermocycler (Applied Biosystem, Foster City, CA). We consider a sample positive if the ct value was ≤ 39 and the melting temperature was between 82°C and 83.8°C

Histopathology

All organs were fixed in 10% neutral formalin and embedded in paraffin and sectioned at

3µm using Leica RM2255 microtome (Leica Microsystems Inc., Bannockburn, IL). The sections were then stained with hematoxylin and eosin and examined using Primo Star

Zeiss light microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

5.4 Results

Clinical signs

Poults were observed twice a day for clinical signs. In both trials and for the two inoculated groups, half of the birds had diarrhea. No other clinical signs were observed. 106

Gross and histopathological lesions

Trial 1. No gross lesions were observed, but a physiological change was detected in one poult of the group 2 at 14 DPI that had some gas in the jejunum; and in one control poult at 7 DPI that had one caecum with yellowish foamy content.

Trial 2. No gross lesions were identified in control poults at all time points. The group 2 at 7 DPI had one bird with yellowish foamy caeca content and some gas in jejunum; and a second bird had normal gut with a pasted vent. The group 3 had intestinal physiological changes at all DPIs. These included caeca with yellowish foamy content, both birds at 18 DPI had pasted vent and one bird at 7 DPI had some gas in the ileum; only one bird at 12 DPI did not have any physiological change.

No histopathological lesions were observed in all organs examined

Weight gain

The weight gain was calculated as the difference between the weight at euthanasia and the weight at the beginning of the experiment. The global weight trend was assessed using a linear equation. In trial 1, the group 1 (slope= 0.0486; R2=0.9031) had better body weight gain compared to the group 2 (slope= 0.0357; R2=0.8278). On the other hand the group 3 had a less predictable behavior as demonstrated by the fact that a second order polynomial and not a linear equation was a better fit for the data (R2= 07425) (Fig.

5.1). In trial 2 the group 1 (slope=0.025; R2=0.9417) had better body weight gain

107 compared to the other two infected groups, which had comparable body weight gain: group 2 (slope=0.0209; R2=0.9742) and group 3 (slope=0.0209; R2=0.8564) (Fig. 5.2).

RT-PCR Astrovirus

Trial 1

The Intestinal content of the poults of the group 2 was positive only at 7 DPI. All organs were tested at 7 and 14 DPI and all were negative. The intestinal content of the poults of the group 3 were positive at all DPI; however, at 21 and 28 DPI the RT-PCR product was very weak indicating a reduction in viral shedding. The bursa, spleen and liver of one bird at 7 DPI was positive, all other organs at all DPI’s were negative (Table 5.1). The intestinal contents of the controls at all DPI’s were negative (data not shown).

Trial 2

The Intestinal content of the poults of the group 2 was positive at all intervals. The bursa and liver of both poults at 7 DPI were positive and in one bird also the pancreas at 7 DPI was positive. All other organs at all DPI were negative. The intestinal content of the poults of the group 3 were positive for both poults at 7 and 12 DPI’s, while at 18 DPI only one poults was positive. However, the RT-PCR product of the intestinal content of one poult at 12 DPI and one at 18 DPI was very weak indicating a reduction in viral shedding. The liver of one bird at 7 DPI was positive; all other organs at all DPI were negative (Table 5.2). The intestinal contents of the controls at all DPI’s were negative

(data not shown).

108

Real-Time PCR parvovirus

Trial 1

The Intestinal contents of the poults of the group 3 were negative at 7 and 14 DPI; and became positive at 21 and 28 DPI. The spleen from one poult at 21 and one at 28 DPI were positive; all other organs at 21 and 28 DPI were negative (Table 5.3). The intestinal contents of the controls at all DPI’s were negative (data not shown).

Trial 2

The Intestinal contents of the poults of the group 3 were negative at 7 DPI; however at 12

DPI one poult was positive and at 18 DPI both poults tested positive. None of the other organs at 12 and 18 DPI were positive (Table 5.4). The intestinal contents of the controls at all DPI’s were negative (data not shown).

5.5 Discussions

Astrovirus and parvovirus have been identified in cases of enteritis in turkeys and chickens. The former has been identified in turkey flocks experiencing enteritis as well as in healthy flocks (Pantin-Jackwood et al., 2008; Pantin-Jackwood et al., 2007; Reynolds and Saif, 1986; Reynolds DL, 1987; Yu M, 2000 ). Moreover, the astrovirus’ pathogenicity for turkeys has been demonstrated in various experimental studies (Jindal et al., 2009a, b; Mor et al., 2011; Nighot et al., 2010; Pantin-Jackwood MJ, 2008;

Spackman et al., 2010; Y. Tang, 2006). The latter has been detected in diseased as well as

109 in healthy flocks (Marusak et al., 2010; Palade, 2011b; Zsak et al., 2009); however its pathogenicity for turkeys has not yet been established.

In our laboratory we attempted to separate astroviruses from parvovirus using serial filtrations or cesium chloride purification, however, due to the small difference in diameter and density between the two viruses, all our attempts were unsuccessful.

Therefore, we decided to use an inoculum containing both astrovirus and parvovirus and compare the pathogenicity of astrovirus alone versus the mixed infection. We performed two trials using SPF turkey poults of different ages to see if the age played a role in the pathogenesis of these viruses. In each trial we randomly divided the poults into three groups: in group 1 the birds were uninoculated, in group 2 they were inoculated with astrovirus alone, while in group 3 they were inoculated with astrovirus and parvovirus. In trial 1, we were able to detect astrovirus in the intestinal content of turkeys of the group 2 only up to 7 DPI and none of the organs tested positive, which suggest a poor astrovirus replication in older birds. A completely different outcome was obtained in the group 3 where the intestinal content was positive for astrovirus at all intervals, although at 21 and

28 DPI we obtained a very weak positive reaction indicating a decrease in shedding. The bursa, spleen and liver of one poult at 7 DPI were positive indicating that viremia had occurred. The intestinal contents were positive for parvovirus at 21 DPI and 28DPI and it seems that once astrovirus replication decreased, as demonstrated by the very weak band detected by RT-PCR, parvovirus replication increased, as demonstrated by Real-Time

PCR detection. These data suggests a competitive effect between the two viruses.

110

In addition, the spleen of one poult at 21 and 28 DPI were positive for parvovirus which might be an indication of viremia. However, since the positivity was at a late cycle, which it is an indication of very low viral load, and that only one out of two birds were positive, further experiments with a higher number of birds and closer intervals are needed to support this hypothesis. In trial 2, as expected, we were able to detect astrovirus in the intestinal contents of turkeys of the group 2 at all DPI. In addition, at 7

DPI, the bursa and the liver of two birds and the pancreas of one bird tested positive for astrovirus, which is an indication of viremia. In the group 3, the intestinal content was positive for astrovirus up to 12 DPI for both poults, but only one poult was positive at 18

DPI, moreover, one liver also tested positive at 7 DPI. Parvovirus followed a similar trend as in trial 1; in fact, we were able to detect it in the intestinal content once the astrovirus shedding decreased. In this trial no organs were positive for parvovirus.

In both experiments, we observed only minimal clinical signs in half of the birds: diarrhea characterized by brownish droppings. The post-mortem examination revealed physiological changes in the intestine in birds of trial 2. Moreover, no lesions were identified by histopathology.

We recorded the weight of each bird before infection and at the time of euthanasia and we observed its trend for all groups. The control group in both trials had better weight gain compared to the other two groups. In trial 1 the body weight of group 3 was not comparable with either groups, while in trial 2, both infected groups showed a similar trend. It was not possible to perform a statistical analysis to compare the body weight gain of controls versus infected groups due to the limited number of birds/group/time

111 point used. Therefore, any speculation regarding the effect of either virus in the stunting syndrome is not possible.

In our study, we saw only minimal clinical sign in the astrovirus infected group and although we detected the virus in the bursa, the histopathology did not show any lesion.

This result may be due to the fact that we sampled at 7 DPI, too late to detect the change in the bursa; in fact, in a previous study this strain produced mild lesions in the bursa at 4

DPI (Y. Tang, 2006).

In conclusion, we observed a potential difference or interference between parvovirus and astrovirus in relation to their replication kinetics. In our experimental settings, the association of the two viruses caused minimal clinical signs and physiological changes in the intestine of 2-4 week-old turkeys. Further studies using different age of birds with a higher numbers of birds to assess different time points after infection are needed to determine the persistence of parvovirus in the spleen and in the bloodstream.

5.6 Acknowledgements

We would like to acknowledge Dr. J. Hanson, K. Berlin, A. Wright, G. Myers for their help with the animal care; Dr. K. Jun for the technical advices in histopathology.

The Salaries and research support was provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University.

112

5.7 References

. Decaesstecker, M., Charlier, G., Meulemans, G., 1986, Significance of parvoviruses, entero-like viruses and reoviruses in the aetiology of the chicken malabsorption syndrome. Avian Pathol 15, 769-782. Domanska-Blicharz, K., Seroka, A., Minta, Z., 2011, One-year molecular survey of astrovirus infection in turkeys in Poland. Arch Virol 156, 1065-1072. Jindal, N., Patnayak, D.P., Ziegler, A.F., Lago, A., Goyal, S.M., 2009a, Duration of growth depression and pathogen shedding in experimentally reproduced poult enteritis syndrome. Avian Dis 53, 517-522. Jindal, N., Patnayak, D.P., Ziegler, A.F., Lago, A., Goyal, S.M., 2009b, Experimental reproduction of poult enteritis syndrome: clinical findings, growth response, and microbiology. Poult Sci 88, 949-958. Jindal N, P.D., Chander Y, Ziegler AF, Goyal SM, 2010, Detection and molecular characterization of enteric viruses in breeder turkeys. Avian Pathology 39, 53-61. Kisary, J., 1985a, Experimental infection of chicken embryos and day-old chickens with parvovirus of chicken origin. Avian Pathol 14, 1-7. Kisary, J., Nagy, B, Bitay, Z., 1984, Presence of parvoviruses in the intestine of chickens showing stunting syndrome. Avian Pathol 13, 339-343. Marusak, R.A., Guy, J.S., Abdul-Aziz, T.A., West, M.A., Fletcher, O.J., Day, J.M., Zsak, L., Barnes, H.J., 2010, Parvovirus-associated cerebellar hypoplasia and hydrocephalus in day old broiler chickens. Avian Dis 54, 156-160. McNulty MS, C.W., McFerran JB, 1980, Detection of astroviruses in turkey faeces by direct electron microscopy. The Veterinary Record 106, 561. Mor, S.K., Abin, M., Costa, G., Durrani, A., Jindal, N., Goyal, S.M., Patnayak, D.P., 2011, The role of type-2 turkey astrovirus in poult enteritis syndrome. Poult Sci 90, 2747-2752. Nighot, P.K., Moeser, A., Ali, R.A., Blikslager, A.T., Koci, M.D., 2010, Astrovirus infection induces sodium malabsorption and redistributes sodium hydrogen exchanger expression. Virology 401, 146-154. Palade, E.A., Kisary, J., Benyeda, Z., Mandoki, M, Balka, G., Jakab, C., Vegh, B., Demeter, Z., Rusvai, M., 2011b, Naturally occurring parvoviral infection in Hungarian broiler flocks. Avian Pathol 40, 191-197. Pantin-Jackwood, M.J., J. M. Day, M. W. Jackwood, and E. Spackman, 2008, Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006. Avian Diseases 52, 235-244. Pantin-Jackwood MJ, S.E., Day JM., 2008, Pathogenesis of type 2 turkey astroviruses with variant capsid genes in 2-day-old specific pathogen free poults. Avian Pathol 37, 193-201. Pantin-Jackwood, M.J., Spackman, E., Day, J.M., Rives, D., 2007, Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms. Avian Dis 51, 674-680.

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Reynolds, D.L., Saif, Y.M., 1986, Astrovirus: a cause of an enteric disease in turkey poults. Avian Dis 30, 728-735. Reynolds DL, S.Y., Theil KW., 1987, A survey of enteric viruses of turkey poults. Avian Diseases 31, 89-98. Saif, L.J.Y., Saif, M., Theil, K. W. , 1985, Enteric Viruses in Diarrheic Turkey Poults. Avian Diseases 29, 798-811. Spackman, E., Day, J.M., Pantin-Jackwood, M.J., 2010, Astrovirus, reovirus, and rotavirus concomitant infection causes decreased weight gain in broad-breasted white poults. Avian Dis 54, 16-21. Tang, Y., Saif, Y.M., 2004, Antigenicity of two turkey astrovirus isolates. Avian Dis 48, 896-901. Trampel, D.W., Kinden, D.A., Solorzano, R.F., Stogsdill, P.L., 1983, Parvovirus-like enteropathy in Missouri turkeys. Avian Dis 27, 49-54. Y. Tang, M.M.I., and Y. M. Saif, 2005, Development of Antigen-Capture Enzyme- Linked Immunosorbent Assay and RT-PCR for Detection of Turkey Astroviruses. AVIAN DISEASES 49, 182-188. Y. Tang, M.V.M., Lucy Ward, and Y. M. Saif, 2006, Pathogenicity of Turkey Astroviruses in Turkey Embryos and Poults. Avian Diseases 50, 526-531. Yu M, I.M., Qureshi MA, Dearth RN, Barnes HJ, Saif YM., 2000 Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus. Avian Dis. 44, 297-304. Zsak, L., Strother, K.O., Day, J.M., 2009, Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses. Avian Dis 53, 83-88.

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Table 5.1 Astrovirus RT-PCR results - Trial 1#

7 DPI 14 DPI 21 DPI 28 DPI

G2 G3 G2 G3 G2 G3 G2 G3

Intestinal Content 2/2* 2/2 0/2 2/2 0/2 2/2 0/2 2/2

Thymus 0/2 0/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Bursa 0/2 1/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Spleen 0/2 1/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Bone Marrow 0/2 0/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Pancreas 0/2 0/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Liver 0/2 1/2 0/2 0/2 N.D. 0/2 N.D. 0/2

Lungs 0/2 0/2 0/2 0/2 N.D. 0/2 N.D. 0/2

*number of RT-PCR positives/number of total samples tested

N.D.= Not done

G2 = group 2; G3 = group 3

#The intestinal contents of the controls were negative at all DPI’s. The other organs of the control group were not tested.

115

Table 5.2 Astrovirus RT-PCR results - Trial 2#

7 DPI 12 DPI 18DPI

G2 G3 G2 G3 G2 G3

Intestinal Content 2/2* 2/2 2/2 2/2 2/2 1/2

Thymus 0/2 0/2 0/2 0/2 0/2 0/2

Bursa 2/2 0/2 0/2 0/2 0/2 0/2

Spleen 0/2 0/2 0/2 0/2 0/2 0/2

Bone Marrow 0/2 0/2 0/2 0/2 0/2 0/2

Pancreas 1/2 0/2 0/2 0/2 0/2 0/2

Liver 2/2 1/2 0/2 0/2 0/2 0/2

Lungs 0/2 0/2 0/2 0/2 0/2 0/2

*number of RT-PCR positives/number of total samples tested

G2 = group 2; G3 = group 3

#The intestinal contents of the controls were negative at all DPI’s. The other organs of the control group were not tested.

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Table 5.3 Parvovirus Real-Time PCR results – Group 3- Trial 1#

7 DPI 14 DPI 21 DPI 28 DPI

Intestinal 0/2* 0/2 2/2 2/2 Content (Ct= 32.96-35.19) (Ct= 34.11-34.94) Thymus N.D. N.D. 0/2 0/2

Bursa N.D. N.D. 0/2 0/2

Spleen N.D. N.D. 1/2 1/2 (Ct= 39.03) (Ct= 37.58) Bone Marrow N.D. N.D. 0/2 0/2

Pancreas N.D. N.D. 0/2 0/2

Liver N.D. N.D. 0/2 0/2

Lungs N.D. N.D. 0/2 0/2

*number of RT-PCR positives/number of total samples tested

N.D.= Not done

#The intestinal contents of the controls were negative at all DPI’s. The other organs of the control group were not tested.

117

Table 5.4 Parvovirus Real-Time PCR results – Group 3- Trial 2#

7 DPI 12 DPI 18 DPI

Intestinal Content 0/2 1/2 2/2 (Ct= 34.73 ) (Ct= 36.09-38.1) Thymus N.D. 0/2 0/2

Bursa N.D. 0/2 0/2

Spleen N.D. 0/2 0/2

Bone Marrow N.D. 0/2 0/2

Pancreas N.D. 0/2 0/2

Liver N.D. 0/2 0/2

Lungs N.D. 0/2 0/2

*number of RT-PCR positives/number of total samples tested

N.D.= Not done

#The intestinal contents of the controls were negative at all DPI’s. The other organs of the control group were not tested.

118

1.6 y = 0.0486x - 0.05 1.4 R² = 0.9031

1.2 y = 0.0357x + 0.15 R² = 0.8278

1 y = 0.0026x2 - 0.0421x + 0.575 R² = 0.7425 0.8

0.6 Group 2 Group 3 0.4 Group 1

Linear (Group 2) 0.2 Poly. (Group 3)

0 Linear (Group 1) 0 5 10 15 20 25 30

Figure 5.1 Trial1- body weight trend assessed with linear equation

119

0.5 y = 0.025x - 0.033 R² = 0.9417 0.45 y = 0.0209x - 0.0622 0.4 R² = 0.9742

0.35 y = 0.0209x - 0.0572 R² = 0.8564 0.3

0.25 Group 1 0.2 Group 2 Group 3 0.15 Linear (Group 1) 0.1 Linear (Group 2) 0.05 Linear (Group 3)

0 0 5 10 15 20

Figure 5.2 Trial2- body weight trend assessed with linear equation

120

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