Studies on Turkey Enteric Viruses: Age Related Prevalence and Impact on Production Performance

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STUDIES ON TURKEY ENTERIC VIRUSES: AGE RELATED PREVALENCE AND IMPACT ON PRODUCTION PERFORMANCE

DISSERTATION

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

Olusegun O. Awe

Graduate Program in Veterinary Preventive Medicine

The Ohio State University

2014

Dissertation Committee:

Dr. C-W. Lee (Advisor)

Dr. Y. M. Saif

Dr. D. J. Jackwood

Dr. G. Rajashekara

Copyrighted by

Olusegun O. Awe

2014

Abstract

The economic impact of viral enteric disease on the poultry industry could be severe and cause devastating losses. Enteric viral disease is characterized by severely delayed growth, lack of uniformity among the flock, lethargy, watery diarrhea, delayed feed consumption, and a decreased feed conversion rate. Enteric viral infections also increase susceptibility to other diseases and prolong the time to market. Enteric viruses tend to predominantly affect young birds. However, the effect of enteric viruses in turkeys older than 4 weeks of age has not been studied in detail. Also, surveillance studies of enteric viruses in turkey flocks have been mainly in young birds and there is limited information about the prevalence of these viruses in older age group of turkeys. Moreover, experimental pathogenesis studies demonstrating the effect of enteric virus in older turkeys are lacking. The objectives of this study were therefore to:

1. Determine the age-related prevalence of enteric viruses in apparently healthy and

diseased turkey flocks

2. Evaluate the in vivo pathogenicity of enteric virus in turkey hens and determine

transmission efficiency of coronavirus

3. Evaluate the age-related susceptibility of turkeys to astrovirus and coronavirus

Fecal samples collected from healthy turkeys from three commercial farms in Ohio from placement to 21 weeks of age, and feces, intestinal contents or litter samples obtained between 2000 and 2010 from four states including North Carolina, Ohio, Pennsylvania and Virginia from 2 to 19-week-old turkeys experiencing enteric disease were used to address the first objective. These samples were tested for adenovirus, astrovirus, coronavirus, parvovirus, reovirus, and rotavirus groups A and D. We found that enteric viruses are widespread in turkeys of wide age range and may also be present throughout the life of the flocks. We also confirmed the presence of rotaviruses in different groups in turkey flocks sampled.

To evaluate the potential effects of these viruses, we carried out pathogenicity testing of enteric viruses in turkeys of different ages using turkey coronavirus and astrovirus. Our results showed that different ages of birds were prone to turkey coronavirus infection, whereas susceptibility to turkey astrovirus decreased with age. Pathogenicity testing in poults showed that turkey coronavirus can cause growth depression and also egg production decline in turkey hens. Neither weight loss nor egg production decline could be attributed to turkey astrovirus. We further evaluated the potential of turkey coronavirus transmission from infected hens to contact hens raised on litter floor. The result showed that turkey coronavirus can efficiently transmit from infected to contact control birds.

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Dedicated to my wife, parents and siblings

Acknowledgments

I am very obliged to my advisor Dr. Chang-Won Lee for his guidance and support throughout my studies. He demonstrated patience in his dealings with me and showed me how to look at everything with a positive attitude. While working in his lab under his supervision I have grown as a person and I will treasure this great experience for all of my life.

I am grateful to my committee members Dr. Yehia M. Saif, Dr. Daral J. Jackwood, and

Dr. G. Rajashekara for their suggestions and comments.

I would like to thank my lab mates Dr. Ahmed Ali, Dr. Kyung-il Kang, Mohamed Elaish and Mohmoud Ibrahim, Dr. Maria Murgia, Dr. Abdul Rauf, and Megan Strother for their technical support and friendship.

I would like to thank Dr. Juliette Hanson, Kingsly Belin, Andrew Wright, Greg Myers and Denis Hartzler for their help with the animal care.

I am very thankful to Mrs. Robin Weimer and Mrs. Hannah Gehman for their kindness and help during all these years.

I am thankful to my wife Foluke Awe and my family for sharing with me this experience.

Vita

July 1999……… …………………………………….Abeokuta Grammar ………………………………………………………..School, Ogun State, Nigeria

July 2007……………………………………………. Doctor of Veterinary Medicine ………………………………………………………..University of Ibadan, Nigeria

October 2007 to August 2009……………………….Veterinary Clinician ……………………………………………………….Ministry of Agriculture, Nigeria

September 2009 to March 2011……………………..Masters of Public Health ……………………………………………………….The Ohio State University

April 2011 to present………………………………..Graduate Research Associate, ………………………………………………………Comparative Veterinary Medicine ………………………………………………………The Ohio State University

Publications

1. Awe, O., Ali, A., Elaish, M., Ibrahim M., Murgia V. M., Saif Y. M. and Lee, C.W. Effect of coronavirus infection on reproductive performance of turkey hens Avian Diseases, 57(3):650-656. 2013. 2. Ali, A., Yassine, H. M., Awe, O., Ibrahim, M., Saif, Y.M. and Lee, C.W. Replication of Swine and Human Influenza Viruses in Juvenile and Layer Turkey Hens. Vet. Microbiol. 2013. 163 (1-2): 71–78.

Fields of Study

Major Field: Veterinary Preventive Medicine

Table of Contents

Abstract……………………………………………………………………………………ii

Acknowledgement………………………………………………………………………...v

Vita…………………………………………………………………………………….....vi

Publications…………………………………………………………………………….....v

Fields of study…………………………………………………………………………….vi

Table of content……………………………………………………………………….....vii

List of tables……………………………………………………………………………..vii

List of figures………………………………………………………………………….....xi

Chapter 1: Literature review……………………………………………………..………..1

1.1 Introduction…………………………………………………………………………..1

1.2 Viral agents implicated in enteric disease……………………………………………4

1.3Transmission of enteric viruses…………………………………………………...... 22

1.4 Diagnostic methods for detection of enteric viruses……………………………...... 23

1.5 Epidemiology of enteric viruses…………………………………………………....29

1.6 Prevention and control……………………………………………………………...31

1.7 References……………………………………………………………………….....33

Chapter 2: Age related prevalence of enteric viruses in healthy and diseased turkey flocks……………………………………………………………………………………..43

2.1Summary……………………………………………………………………………..43

2.2 Introduction………………………………………………………………………….44

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2.3. Materials and Methods……………………………………………………………....46

2.4 Results……………………………………………………………………………...... 49

2.5 Discussion…………………………………………………………………………....53

2.6 Acknowledgement…………………………………………………………………...56

2.6 References…………………………………………………………………………....57

Chapter 3: Effect of coronavirus on egg production in turkey hens……………………..68

3.1Summary…………………………………………………………………………...... 68

3.2Introduction………………………………………………………………………...... 69

3.3. Materials and Methods………………………………………………………………71

3.4 Results………………………………………………………………………………..74

3.5 Discussion……………………………………………………………………………78

3.6 Acknowledgement…………………………………………………………………...82

3.7 References……………………………………………………………………………83

Chapter 4: Age related susceptibility of turkeys to enteric viruses……………………...91

4.1 Summary…………………………………………………………………………...... 91

4.2 Introduction………………………………………………………………………...... 92

4.3. Materials and Methods……………………………………………………………....93

4.4. Results……………………………………………………………………………....98

4.5. Discussion……………………………………………………………………….....101

4.6. Acknowledgement…………………………………………………………………104

4.7. References……………………………………………………………………….....105

Bibliography……………………………………………………………………………114

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List of Tables

Table 2.1 Summary of enteric viruses analyzed in turkey flocks...... ….59

Table 2.2 Summary of enteric viruses from healthy turkey flock……………………….60

Table 2.3 Summary of enteric viruses from diseased turkey flock………………………61

Table 2.4 Summary of detected viruses from field samples……………………………..62

Table 3.1 Virus detection by RT-PCR in cloacal swabs, jejunum and ileum of TCoV infected and contact control turkey hens ...... 86

Table 4.1 Comparison of RT-PCR and virus isolation for detection of TAstV in experimentally infected turkey cloacal swabs………………………………………….107

Table 4.2 Comparison of RT-PCR and virus isolation for detection of TCoV in experimentally infected turkey cloacal swabs………………………………………….108

Table 4.3 Virus detection by RT-PCR in cloacal swabs of 30- and 52-week-old turkeys in trials 1 and 2………………………………………………………………………...... 109

Table 4.4 Virus detection in cloacal swabs, intestinal contents and tissues of 30-week-old turkeys in trial 3…………………………………………………………………...... 110

List of Figures

Figure 2.1. Prevalence of enteric viruses by age in healthy and disease flocks…………63

Figure 2.2. Prevalence of rotavirruses in healthy and diseased turkey flock ...... 64

Figure 2.3. Phylogenetic tree of VP6 gene of rotavirus ...... 65

Figure 2.4. Phylogenetic tree of VP4 gene of rotavirus ...... 66

Figure 2.5. Phylogenetic tree of VP7 gene of rotavirus ...... 67

Figure 3.1. Egg production graph...... 87

Figure 3.2. Pathological changes produced by coronavirus in turkey hens ...... 88

Figure 3.3. Histopathological lesions produced by coronavirus n turkey hens ...... 89

Figure 3.4. Turkey coronavirus detection by RT-PCR in cloacal swabs, intestinal contents and reproductive tissues of infected turkey……………………………………90

Figure 4.1 Body weight graph of turkey poults………………………………………..111

Figure 4.2 Egg production graphs………………………………………….... ………..112

Figure 4.3 Turkey astrovirus serum antibody detection in different ages of turkeys….113

Chapter 1: Literature Review

1.1 Introduction

The ability of the turkey to achieve its genetic potential in relation to growth and feed utilization correlates with the health of the intestinal tract. The gastrointestinal (GI) tract is the most extensively exposed surface in the body and is continuously open to wide range of infections. The GI tract infections occur commonly in turkeys of different ages although it tend to predominate in young birds. Infectious diseases affecting the digestive tract of commercial poultry are proposed to result in more economic loss than those affecting any other system as the effects of enteric diseases continue long after recovery.

1.1.1 Poult Enteritis Complex (PEC). PEC is a general term used to denote all infectious intestinal disease of young turkeys (Barnes et al. 2000). It encompasses a group of multifactorial, transmissible and infectious disease of turkeys with major impact in turkeys younger than six weeks of age (Spackman et al. 2005). It incorporates a number of described diseases and clinical enteric infections of uncertain etiology as there is paucity of knowledge about the causes, and interactions of enteric pathogens and

1 opportunistic infections in young turkeys (Barnes et al. 2000). The primary disease presentation of PEC is diarrhea, restlessness and a general poor condition of the poults with significant economic losses in the turkey industry, because the full growth potential of the turkeys cannot be reached due to stunting and decreased weight gain (Spackman et al. 2005).

The clinical manifestation of PEC in turkeys is similar to another disease in chickens commonly referred to as runting and stunting syndrome, and although the relationship between both disease has not been clarified, evidence suggests that the two are distinct syndromes (Shapiro et al. 1998). PEC was reported to be a major concern in the southeastern United States (U.S.) where an estimated 60 - 90 percent of all flocks experienced PEC related symptoms, and similar disease conditions have been reported from most regions where turkeys are commercially raised (Barnes et al. 2000). Different terms such as coronaviral enteritis, malabsorption syndrome, maldigestion syndrome, poult enteritis and mortality syndrome (PEMS), spiking mortality of turkeys and turkey viral enteritis have been used to describe different syndromes of enteric disease, and all are grouped under the PEC (Jindal et al. 2010). When PEC is coupled with mortality the disease is classified as PEMS (Barnes et al. 2000). The later manifestation could be confusing because mortality can occur when PEC is encountered, hence this term will not be used. PEC is an economically devastating disease that affects turkeys, and was first identified in high density turkey producing areas of the southeastern U.S. in 1991

(Doerfler et al. 2007). It was initially contained in North Carolina, South Carolina and

Georgia till 1994, it was however confirmed as a disease in turkeys in several other states

2 by 1995 (Doerfler et al. 2007). It is an acute, infectious and transmissible intestinal disease characterized by high morbidity (Barnes et al. 1996). Poults presenting with this form of disease develop agitation, high-pitched vocalization and continuous movement.

Birds become anorexic and act in a way that suggest feed or water intake irritates their mouth or upper gastrointestinal tract (Doerfler et al. 2007). While some flocks completely refuse to eat, others may consume litter and peck at the feed trying to sort out larger particles. This drop in feed consumption may initially be compensated by increased water intake. However, water intake also declines in parallel with reduced feed intake. Severe diarrhea develops in association with dehydration which results from the osmotic effects of undigested feed and unabsorbed feed (Barnes et al. 2000). Size variation among birds in the flocks is observed as weight loss set in within few days, and lack of uniformity is more evident by a week after the onset of clinical symptoms (Barnes et al. 2000). It was readily reproduced by inoculating young poults with feces or intestinal homogenates (Reynolds et al. 1986), or by placing susceptible birds in contact with litter from an affected flock (Yu et al. 2000).

It is identified by its characteristic mortality pattern; mortality may range between

25 and 96% in field cases (Doerfler et al. 2007), in severe outbreaks, infected turkeys may exhibit mortality that exceeds 50% between 1 and 4 weeks of age (Barnes et al.

2000). Furthermore, birds that survive are stunted by 40% or more and never attain expected market weights at the 18 to 20 week of age (Doerfler et al. 2007). Initial attempts to characterize the condition were curtailed since it was not known if it is a single disease or a complex of several diseases. There are two primary clinical forms of

3 the disease that have been recognized – the milder form called excess mortality of turkeys

(EMT) and the more severe form called spiking mortality of turkeys (SMT) (Carver et al.

2001). SMT occurs when flock mortality equals or exceed 1% for three or more consecutive days or a total mortality in excess of 9% for a three week period (Barnes et al. 1996). In addition, affected birds are immune-compromised, coupled with a variety of physiological abnormalities including reduced body temperatures, reduced energy metabolism and hypothyroidism (Doerfler et al. 1998). This particular virulent enteric disease characterized by a sharp peak of mortality emerged in 1991 in dense turkey production areas of western North Carolina, and was initially named SMT because of its associated mortality patterns, but was later changed to PEMS following the discovery of a milder clinical form of the disease in 1994 (Barnes et al. 2000). In cases of EMT, mortality does not go beyond 1% per day for three or more consecutive days, and overall mortality vary between 2 to 9% over a three week period (Carver et al. 2001).

1.2 Viral agents implicated in enteric disease

Viruses are a major group of pathogens affecting the GI tract of young turkeys, and consequent infections could affect the major functions of the GI tract including absorption, secretion, and regeneration and probably resulting in severe and protracted enteric disease. Viral enteritis is common in young turkeys, and several viruses have been linked with disease. Infections with a single virus are typically mild but combined viral infections which are more frequently observed in poults may lead to prominent symptoms and extensive lesions spanning through several regions of the GI tract. The replication

4 sites of viruses on the surface of the villus vary (Pantin-Jackwood et al. 2008b, Spackman et al. 2010) between viruses and vulnerability to opportunistic infections is typically increased as a result of viral infections from damage to the integrity of the GI tract and exposure of sites essential for replication of secondary pathogens.

Several different viruses including adenovirus, astrovirus, coronavirus, parvovirus, reovirus, and rotavirus have been implicated as important causes of gastrointestinal tract infections in turkeys (Guy et al. 1998). Different viral combinations result in different clinical syndromes and a wide variety of outcomes ranging from inapparent economically insignificant effects to those that are severe and economically devastating. The outcome of these infections is dependent on a number of interacting factors including the age and immune status of the affected birds and also the virulence of the viruses involved. Because other factors in field situations such as management, nutrition and environmental conditions complicate these infections, it is often difficult to assess the true role of viruses in naturally occurring gastrointestinal diseases of turkeys

(Spackman et al. 2010).

1.2.1 Rotavirus. Rotaviruses were identified in 1977 and have been associated with enteritis of variable severity in turkeys during the early stages of life (Bergland et al.

1977). Rotaviruses are classified into the genus Rotavirus within the family Reoviridae

(Estes et al. 1990), and are non-enveloped particles with a diameter of 70-100nm with a genome consisting of 11 double-stranded RNA segments that codes for six viral structural proteins and five or six nonstructural proteins. The virus particles consist of

5 three protein layers; the outer layer of the virus particle is formed by VP4 which is made of up two portions VP5 and VP8 which represents products cleaved by trypsin, together with VP7 which contain serotype-specific epitopes (Kapikian et al. 2007). The intermediate layer is made up of the viral structural protein VP6 which is most conserved protein and is used for serological grouping of rotaviruses. The inner layer is formed by

VP2 surrounding a complex of VP1 and VP3, which are the RNA-dependent RNA polymerase and guanylyl transferase, respectively. At least five nonstructural proteins are encoded by the rotavirus genome with diverse functions. NSP1 is responsible for modulation of host immune response and the NSP3 regulates viral gene expression while the NSP4 gene is responsible for induction of diarrhea (Ball et al. 1996).

Classification of avian rotaviruses was initially based on immunofluorescent antibody assays and polyacrlylamide gel electrophoresis (PAGE) analysis of double stranded RNA segments (McNulty et al. 1980). Turkey rotaviruses were shown to share a common antigen with mammalian group A rotaviruses (McNulty et al. 1980), however, rotaviruses that are antigenically distinct from group A turkey rotaviruses were also detected from poults in the U.S. (Saif et al. 1985). These antigenically distinct turkey rotaviruses were referred to as rotavirus-like particles viruses (RVLVs), and its 11 double stranded RNA segments produced distinct genome electropherotype different from those of group A rotaviruses in polyacrylamide gels (Saif et al. 1985). Subsequently, additional turkey rotaviruses that possess novel genome electropherotype that appears to be antigenically distinct from group A rotaviruses and RVLVs were detected and were termed atypical rotaviruses (Saif et al. 1985).

The symptoms of disease include diarrhea and depression, increased mortality and chronic runting and stunting characterized by weight loss have also been associated with rotavirus infections. Experimental inoculation of turkeys with rotavirus results in mild to inapparent infection and the variation in severity of infection may be attributed to differing virulence of rotavirus strains or the interaction of other pathogens, environmental and/or management factors (McNulty et al. 1997). Although limited information about the pathogenesis of avian rotavirus infection exists, one study demonstrated viral antigen staining following rotavirus infection in the enterocytes in distal portion of the intestinal villi (Spackman et al. 2010). Rotavirus shedding was detected in all inoculated poults up to 10 days post infection (DPI) with proportion of poults shedding decreasing to about 60 percent at 2 weeks post inoculation (Spackman et al. 2010).

Surveillance studies in the 1980s in the U.S. revealed that RVLVs were detected less frequently in apparently healthy than in diseased flocks, however, rotaviruses A were detected more frequently in normal than in diseased flock (Reynolds et al. 1987). This raises speculation as to the pathogenic effect rotaviruses may have on poults in the field.

Although commercial turkeys are often infected during the first weeks of life with rotaviruses, the epidemiology of different groups of rotaviruses detected appear to follow an age related pattern. RVLV and atypical rotaviruses were never detected in commercial turkeys less than 10 days old whereas only group A rotavirus was detected in commercial turkeys less than 7 days old (Theil et al. 1987).

Studies on the occurrence of rotaviruses in birds were also carried out based on the use of PAGE (Trojnar et al. 2010, Otto et al. 2012). The rotaviruses that infect birds were reported to belong to groups A, D, F and G (Otto et al. 2012). Further studies from

India, Germany (Otto et al. 2006) and Bangladesh differentiated rotaviruses into groups and found that group D rotaviruses were the most widespread rotaviruses in diarrheic turkey poults. Furthermore, avian rotaviruses of group F and G have been detected occasionally (Otto et al. 2012). However, classification of rotaviruses on the basis of

RNA migration pattern is problematic due to the possible occurrence of genome rearrangement (Desselberger et al. 1996). In contrast to group A rotaviruses, other rotaviruses detected in birds could not be adapted to cell culture thus hindering further characterization. Enzyme linked immunosorbent assay (ELISA) were also used to detect rotaviruses but because of limited availability of antisera, reverse transcriptase - polymerase chain reaction (RT-PCR) have been employed and has aided sequence based classification for genotyping of rotaviruses (Schumann et al. 2009, Trojnar et al. 2010,

Otto et al. 2012). Unfortunately, additional surveillance or pathogenesis studies investigating the roles of different rotavirus serogroups on enteric health of U.S. poultry are lacking. Recent surveillance studies utilized a universal NSP4-based PCR approach which detects all avian rotaviruses (Pantin-Jackwood et al. 2007, Pantin-Jackwood et al.

2008a, Jindal et al. 2010a), and efforts geared towards differentiation of rotaviruses into serogroups were not made.

1.2.2 Astrovirus. Astroviruses were first identified in 1980 from intestinal contents of turkey poults with diarrhea and increased mortality (McNulty et al. 1980). Afterwards, the widespread occurrence of astrovirus infection in U.S. turkeys has been documented since 1985 (Reynolds et al. 1987, Yu et al. 2000, Sellers et al. 2004a). Avian astrovirus belong to the genus Avastrovirus in the family Astroviridae, and are small, nonenveloped, positive sense RNA viruses of 28 to 30 nm in diameter with a characteristic star shaped morphology from which their name was derived (Matsui et al.

2001). Their viral genome is 6.8 to 7.9 kilobases in length and contains 3 open reading frames which encode nonstructural proteins (ORF1a), viral RNA-dependent RNA polymerase (ORFIb), and the capsid protein (ORF2) (Jiang et al. 1993). The prototype astrovirus strain, turkey astrovirus 1 (TAstV-1) was first identified in the U.S. in 1985

(Saif et al. 1985), and a second turkey astrovirus distinct antigenically and genetically from TAstV-1 was later discovered and was designated as turkey astrovirus type 2

(TAstV-2) (Koci et al. 2000). The TAstV-2 is commonly found and distinct subtypes were also identified with differences in antigenicity previously reported.

Clinical signs of disease somewhat vary but generally include diarrhea, lethargy, litter eating and nervousness. Mild to moderate disease severity is observed with negligible mortality and morbidity that manifests as growth depression is of major concern (Reynolds et al. 1986). In experimental infections, turkeys infected with TAstV-

2 developed watery diarrhea which was observed from 2 DPI and continued through 12

DPI (Reynolds et al, 1986, Koci et al. 2003). Gross pathology observed in infected birds includes dilated ceca containing yellowish frothy contents, fluid distention of the

9 intestines, and loss of tone and hyperemia with intestines of infected poults appearing 3 to 5 times larger than controls (Behling-Kelly et al. 2002). In another study, astrovirus infection manifested as growth depression and infected birds had significantly lower body weights when compared to control groups which were attributed to decreased absorption of nutrients in infected birds (Koci et al. 2003). Astrovirus infection in specific pathogen free (SPF) turkey poults also caused a significant decrease in intestinal

D-xylose absorption (Ismail et al. 2003). Decreased intestinal maltase absorption was also reported in commercial flocks inoculated with astrovirus resulting in maldigestion of disaccharides, malabsorption and the consequent osmotic diarrhea (Thouvenelle et al.

1995a).

Microscopic lesions following astrovirus infection include mild crypt hyperplasia which results in increased crypt depth with histological changes being prominent in the proximal jejunum one day after infection, and all parts of the small intestine affected by 5

DPI (Thouvenelle et al. 1995b). Astrovirus infection does not induce villous atrophy; however, mild shortening of the villi coupled with clusters of necrotic enterocytes along villi bases has been reported. Replication of astrovirus other than in the intestines has also been reported. Astrovirus was isolated from thymus, bursa, spleen, kidney, pancreas and plasma (Koci et al. 2003). Lymphoid depletion occurring in bursa as a result of astrovirus infection suggest a possible effect on the immune system and could be a result of apoptosis indirectly induced by astrovirus on the bursal epithelial cells (Qureshi et al.

2000).

The principal site of astrovirus replication was reported to be in the cytoplasm of matured villous epithelial cells in the jejunum and less commonly in the epithelial cells of other intestinal sections (Pantin-Jackwood et al. 2008b). Viral replication was also detected in cells in the crypts and although viremia may occur following infection with turkey astrovirus, only intestinal epithelial cells appear to support virus replication

(Pantin-Jackwood et al. 2008b). Following experimental infection of poults, high proportion of birds shed astrovirus till 10 DPI. However, proportion of shedding decreased to 40 percent 2 weeks post inoculation (Spackman et al. 2010).

Astrovirus infections are ubiquitous in turkey populations and have been isolated from commercial turkey poults experiencing viral enteritis (Reynolds et al. 1986, Qureshi et al. 2000). Disease manifestation are commonly seen between 1 and 3 weeks of age and typically last 10-14 days (Reynolds et al. 1986). Astrovirus has also been demonstrated in apparently health turkey flocks and longitudinal studies identified the virus as the most prevalent enteric virus in normal commercial flocks (Pantin-Jackwood et al. 2008a, Jindal et al. 2010b). Astrovirus infections usually occur in the first 4 weeks of life and are rarely detected alone and it is not uncommon to observe multiple enteric viruses in varying combination in cases of enteric disease. Frequently, co-infection of astrovirus and group D rotaviruses was reported in the 1980’s (Reynolds et al. 1987) and although rotaviruses were not classified into groups, it’s co-infection with astrovirus was also observed in recent studies (Pantin-Jackwood et al. 2007).

1.2.3 Parvovirus. Parvoviruses belong to the family Parvoviridae which has two subfamilies, Densovirinae and Parvovirinae. Chicken and turkey parvoviruses are grouped under the genus Parvovirus in the subfamily Parvovirinae (ICTV 2012).

Parvovirus possess a single stranded DNA genome which is about 5 – 6 kilobases in length. They are nonenveloped with an icosahedral capsid of about 18 to 26 nm in diameter (Berns et al. 1990). Replication of parvovirus depends on cellular factors that are found in cells during the S phase of the cell cycle, therefore its replication and pathogenic effects occur mainly in cells with a high rate of cellular proliferation (Berns et al. 1990). Intestinal disease result from parvovirus infection of epithelial cells in the crypts of Leberkuhns and infection of the crypt cells and intestinal germinal epithelium lead to impaired replacement of villus absorptive cells that are shed at villus tips (Guy

1998). This impairment results from atrophy of the villus and the consequent malabsorption and diarrhea (Moon et al. 1978). Experimental infection of poults with a combination of astrovirus and parvovirus revealed a potential interference of two enteric viruses in turkeys. A clear pattern of viral replication of two viruses was observed, astrovirus was detected at early time points up to 14 DPI and parvovirus was detected in the latter time points after 12 DPI (Murgia 2012).

Although parvovirus has been identified in birds exhibiting signs of enteric disease, they have also been observed in healthy flocks (Trampel et al. 1983, Zsak et al.

2008, Zsak et al. 2009, Palade et al. 2011a, Palade et al. 2011b) and their role in the enteric syndromes of poultry remains unclear. Chicken parvovirus induced growth retardation following experimental infection in 2-day-old broiler chickens (Zsak et al.

2013). Experimental infections with a chicken origin parvovirus also revealed growth depression in commercial birds as compared to controls (Kisary et al. 1985). However, this was not a consistent finding as another group of researchers were unable to reproduce the stunting in commercial broilers using the same isolate (Decaesstecker et al. 1986).

Conversely, there was no effect on weight loss in the SPF white leghorn chickens (Kisary et al. 1985).

Parvovirus is widely distributed in the U.S. and in Europe. Surveillance studies of

U.S. chicken and turkeys of unknown health status between 2 and 7 weeks of age revealed a prevalence of 77% and 78%, respectively (Zsak et al. 2009). An overall prevalence of 71% was observed in turkeys with symptoms of enteric disease. In addition, a higher prevalence (80%) was observed in birds 8 and 19 weeks of age as compared to 60% prevalence in birds between 1 and 7 weeks of age (Murgia et al. 2012).

Parvoviruses have also been detected in healthy chicken and turkey flocks in Hungary

(Palade et al. 2011a) and 47% prevalence was reported from birds with disease (Palade et al. 2011b). In addition, a survey of poultry flocks affected by enteritis in Croatia revealed parvovirus infection in 7 out of 9 chicken flocks and in 1 out of 6 turkey flocks (Bidin et al. 2011). Phylogenetic analysis of majority of chicken and turkey parvoviruses based on the nonstructural gene showed clustering in separate branches, which suggest species specific adaptation (Murgia et al. 2012). However, some turkey strains have been shown to be closely related to chicken parvovirus indicating a possibility of interspecies transmission between chickens and turkeys (Domanska-Blicharz et al. 2012, Murgia et al.

2012).

1.2.4 Adenovirus. Adenoviruses are common infectious agent in poultry and also in wild birds and are non-enveloped viruses with 70 to 90 nm in diameter and possess a linear, double stranded DNA genome (Beach et al. 2009). Four genera have been recognized based on phylogenetic differences. Mastadenoviruses infect a wide variety of mammalian species including humans, while adenoviruses that infect a wide range of avian species belong to the genus aviadenoviruses (Beach et al. 2009). Atadenovirus and Siadenovirus are adenoviruses which differ in genetic structure and evolutionary history from mastadenoviruses and aviadenoviruses (Benkö et al. 1998). The genus Siadenovirus contain viruses that are speculated to have originated from amphibians and then adapted to avian species (Davison et al. 2003). The genus derived its name from its specific open reading frame (ORF) whose gene product is high in sequence identity with bacterial sialidase proteins (Davison et al. 2000). Frog adenovirus 1, marble spleen disease virus, and turkey hemorrhagic enteritis virus (THEV), the cause of acute virus disease of turkeys, are the recognized members of this genus.

Hemorrhagic enteritis virus is the cause of an economically important acute viral disease of turkeys 4 weeks of age and older (Guy et al. 1998). It occurs in majority of turkey producing areas of the world including Canada, England, Germany, Australia,

India, Japan, Israel, and the U.S. (Guy et al. 1998). The disease was first reported in

Minnesota and in at least 10 other states, occurring in confinement and free range turkeys with a tendency to infect subsequent flocks placed on the same premises (Hofstad et al.

1984). Although serologic evidence indicates a high incidence of THEV infection in

14 turkeys, the low incidence of clinical disease is believed to be due to the presence of avirulent or low virulent strains in turkey populations (Beach et al. 2009). Financial losses from hemorrhagic enteritis in the U.S. were reported to have exceeded $3 million in the 1980’s (Hofstad et al. 1984), however currently with the extensive use of vaccines highly pathogenic outbreaks are rare.

Replication of hemorrhagic enteritis virus occurs in cells of the reticuloendothelial system and the spleen appears to be the major site of virus replication (Hofstad 1984).

The mechanism by which hemorrhagic enteric virus causes intestinal disease and hemorrhage is inconclusive (Guy et al. 1998). In contrast to other intestinal viruses,

THEV does not replicate in intestinal epithelium, and different mechanism suggesting replication in intestinal endothelial cells resulting in vascular damage and ischemic necrosis of intestinal villi was proposed (Beinfield et al. 1990).

Clinical signs associated with THEV infection are most commonly observed in 4- to 12-wk-old turkeys and disease in affected flocks for 7-10 days (Guy et al. 1998). Birds exhibit rapid depression, bloody droppings, and sudden death, and feces containing bloody discharge are often seen on the skin and feathers near the vents of lethargic and dead birds (Fitzgerald et al. 2013). Signs of disease tend to subside within 6–10 days of the appearance of bloody droppings in naturally infected flocks (Fitzgerald et al. 2013).

Mortality in field outbreaks ranges from 1 to 60% and reaches approximately 80% in experimentally inoculated birds (Guy et al. 1998).

Gross pathology is seen mainly in spleen and intestines. Spleens are enlarged and mottled and the small intestines are commonly distended, congested, and filled with

15 bloody exudates (Guy et al. 1998). Intestinal lesions are more prominent in the proximal small intestine but may extend to other distal portions in severe cases (Fitzgerald et al.

2013). Microscopically, characteristic lesions of the disease are seen in immune and gastrointestinal tract (Guy et al. 1998). Hyperplasia of white pulp and lymphoid necrosis is observed in the spleen at death (Fitzgerald et al. 2013). Intestinal lesions include severely congested mucosa, degeneration and sloughing of villus epithelium, and hemorrhages at villus tips as a result of endothelial disruption.

1.2.5 Reovirus. Avian reoviruses (ARV) have been associated with enteric disease in poultry and they are also involved in other diseases such as viral arthritis/tenosynovitis, respiratory disease, and malabsorption syndrome (Rosenberger et al. 2003). The causal relationship of ARV to these diseases remains unproven with the exception of viral arthritis/tenosynovitis (Guy et al. 1998). Reoviruses are prevalent in poultry worldwide, and have been isolated from fecal samples and tissues of clinically normal and diseased birds (Gershowitz et al. 1973).

ARV belong to Orthoreovirus genus in the family Reoviridae and are nonenveloped virus with icosahedral capsid shells and a particle size of 70-80 nm

(Spandidos et al. 1976). The genome contains linear double stranded RNA with 10 segments which are grouped into large (L1, L2, L3), medium (M1, M2, M3), and small

(S1, S2, S3, S4) based on migration pattern on polyacrylamide gel electrophoresis

(Varela et al. 1994). The segmented genome encodes for eight structural and four nonstructural proteins as follows: 3 λ proteins (λA, λB, and λC) by L segments, two μ

16 proteins (μA and μB) by M segments, and three σ proteins (σC, σA, and σB) by S segments (Varela et al. 1994, Varela et al. 1996). The M3 and S4 segments encode two major nonstructural proteins μNS and σNS, respectively, while the S1 segment encodes p10 and p17 (Shmulevitz et al. 2002).

Pathogenic and nonpathogenic strains of ARV exist with majority of them being nonpathogenic (Jones et al. 2000). Reoviruses have been isolated from chicken and turkeys and also from other avian species including ducks and pigeons with diarrhea, and gray parrots and quail with enteritis (McFerran et al. 1976, Ritter et al. 1986). Clinical disease caused by ARV has been well described and has a variety of presentations, including viral arthritis, respiratory disease, immunosuppression, malabsorption with stunting, and subclinical infection (Cook et al. 1984a, Cook et al. 1984b). Clinical disease in turkeys has been reported to be similar to what is observed in chickens, and includes viral arthritis (Page et al. 1982) and poult enteritis (Reynolds et al. 1987). It was previously assumed that all ARV from chickens and turkeys are closely related, however, a group of ARV that are genetically distinct from chicken origin reoviruses have been collected from commercial turkey flocks with enteric disease (Sellers et al. 2004b).

Turkey enteric reoviruses have been isolated from intestines of healthy turkeys and also from commercial turkeys with enteric diseases (Dees et al. 1972). Experimental studies in poults showed that turkey reovirus are probably not a primary cause of poult enteritis as demonstrated by minimal clinical signs, absence of gross lesions and mild microscopic lesions in the intestines of inoculated commercial and SPF poults (Spackman et al. 2005).

In addition, there are conflicting reports about reovirus replication in the intestinal

17 tissues. In one study, reovirus was reported to replicate poorly as evident by rare detection of virus in the intestines following experimentally inoculated poults (Spackman et al. 2005). This differs from another report that showed that reovirus replicated very well in the intestines (Simmons et al. 1972), which may indicate strain differences as the isolate used in this study shared a low nucleotide identity of 45 – 47% with recently published reovirus strains.

Although reoviruses may not be primary pathogens in the development of enteric disease, the severe bursa atrophy caused by reovirus in poults infected at young age may lead to transient and possibly permanent immunosuppression (Spackman et al. 2005b).

Therefore induction of immunosuppression at an early age may increase susceptibility to opportunistic pathogens exacerbating the clinical enteric disease. Turkey reovirus infection has also been associated with decreased body weight gain; this growth depression is consistent with their etiological role in stunting, runting and malabsorption

(Rosenberger et al. 2003).

1.2.6 Coronavirus. Turkey coronavirus (TCoV) is the etiology of an acute infectious enteric disease of turkeys characterized by depression, anorexia, diarrhea, and decreased weight gain. This severe enteric disease of turkeys is known by different names including

Bluecomb disease, mud fever, transmissible enteritis, and coronaviral enteritis.

Coronaviruses are large, enveloped, nonsegmented, positive-sense single-stranded

RNA viruses belonging to the family Coronaviridae (Cavanagh et al. 2001). Their genome is approximately 30 kb long from which they transcribe a set of multiple 3’-

18 coterminal nested subgenomic mRNA (Sawicki et al. 2007). The virus possesses roughly spherical, pleomorphic virions with diameters ranging from 50–200 nm with characteristic petal-shaped spikes on their surface that are responsible for their crown- shaped morphologic appearance as observed using an electron microscope (Dea et al.

1986). The virus particle is made up of four major structural proteins including the highly variable spike (S) glycoprotein, the conserved membrane (M), nucleocapsid (N), and small envelope (E) proteins. Coronaviruses that have been detected in poultry belong to the genus Gammacoronavirus, and consists of infectious bronchitis virus (IBV) of chicken and TCoV (de Groot et al. 2008).

In 1951, an enteric disease of turkeys was described and was initially named mud fever (Peterson et al. 1951). Because of clinical disease manifestation similar to avian monocytosis (bluecomb disease of chickens), the disease was later named as bluecomb disease, and resulted in severe economic losses in North America in the 1950s and 1960s

(Nagaraja et al. 1997). Economic loss due to the disease were ascribed to increased mortality and weight loss in affected turkeys, and was regarded as the most costly disease plaguing Minnesota turkey production between 1951 and 1971 (Nagaraja et al. 1997).

Because the disease resulted in about 23% mortality in Minnesota turkey flocks, effort were geared towards identifying the etiology of bluecomb (Patel et al. 1977). Although different pathogens including reoviruses, enteroviruses, and Campylobacter spp. were recognized in affected turkeys, attempts to experimentally reproduce the disease with the identified agents were futile (Tumlin et al. 1958, Truscott et al. 1968, Deshmukh et al.

1969, Fujisaki et al. 1969, Wooley et al. 1972).

Propagation of a virus from bluecomb disease-affected turkeys in embryonated chicken and turkey eggs was successful in 1971 (Adams et al. 1971). A coronavirus was identified as the etiology in 1973 (Panigrahy et al. 1973, Ritchie et al. 1973) coupled with experimental reproduction of the disease. Following extensive depopulation and decontamination effort in 1971, the disease and the virus apparently disappeared. In the last decade, the virus was sporadically detected in turkey flocks in Indiana, North

Carolina, and Georgia, and associated as a cause of PEMS characterized by high mortality and severe growth depression (Barnes et al. 2000).

TCoV replicates in enterocytes at the apical portion of the intestinal villi in the jejunum and ileum, and also in the epithelium of bursa of Fabricius (Guy et al. 2002).

TCoV could be detected using reverse transcriptase - polymerase chain reaction (RT-

PCR) as early as 1 DPI in 2-day-old poults. Although viral shedding persists till 14 DPI, none of the cloacal swabs from the birds was positive at 21 DPI (Gomaa et al. 2009). In

4-week old birds, shedding also continued up to 14 DPI, but only one fecal swab was positive at 16 DPI suggesting that the duration and course of TCoV infection does not differ between age of infection (Gomaa et al. 2009). In another study, TCoV was shed up to 7 weeks after inoculation using another strain, TCoV NC95 isolate (Breslin et al.

2000). Protective immunity was acquired after TCoV infection as attempt at reinfecting poults previously exposed to TCoV resulted in no resumption of clinical symptoms

(Gomaa et al. 2009).

Gross lesions are found mainly in the intestines and bursa of Fabricius. The duodenum and jejunum are pale, and distended with watery, gaseous contents. The ceca

20 are also distended and filled with watery contents (Guy et al. 2013). Atrophy of the bursa of Fabricius may also be observed. Microscopic lesions are present in the intestinal villous epithelium and the epithelium of the bursa of Fabricius. Villus atrophy is evident in the intestines along with increased numbers of mononuclear inflammatory cells in the lamina propria, decreased numbers of goblet cells on villous tips, and loss of microvilli

(Adams et al. 1970). In the bursa, moderate lymphoid atrophy of follicular cells and intense heterophilic infiltration are observed (Guy et al. 2000)

Clinical disease is commonly observed in young turkeys during the first few weeks of life, and following a short incubation period of 1 to 3 days affected birds develop depression, watery diarrhea, loss of weight and dehydration. Morbidity is usually about 100% but with variable mortality which ranges from 5 to 50% (Guy et al. 1998). In field cases, clinical signs occur suddenly usually with high morbidity. Birds exhibit depression, anorexia, and decreased water consumption, watery diarrhea, and dehydration, hypothermia and weight loss. Fecal droppings are watery and frothy, green to brownish in color and may contain mucus and urates. Increased mortality, growth depression, and poor feed conversion are shown by flocks experiencing TCoV infection compared to uninfected flocks (Rives et al. 1998). Mortality varies in flocks with disease, but may be high depending on the age of the birds, multiple infection, management practices and environmental conditions. In breeder hens, TCoVinfection has been reported to results in a rapid drop in egg production (Nagaraja et al. 1997). The quality of eggs produced is also affected as hens produce white, chalky eggs that lack normal pigmentation (Nagaraja et al. 1997). Although TCoV has been identified as causative

21 agent of transmissible enteritis in most turkey producing regions of the U.S., sporadic outbreaks in recent years were reported in North Carolina (Tilley et al. 2013) )and

Arkansas (Wooming et al. 2013).

1.3 Transmission

Enteric virus infection is mediated via fecal-oral route and transmission of pathogens appears to require physical contact with infected fecal material (Doerfler et al. 2007).

Infected birds excrete large amounts of virus in their feces resulting in rapid spread of infection by direct and indirect contact. Enteric viruses are excreted in avian feces in very large numbers (Yason et al. 1987) and are shed in feces of infected birds and spread horizontally through ingestion of feces and feces-contaminated materials. Enteric viruses can be shed in droppings of turkeys for several weeks after recovery from clinical disease

(Breslin et al. 2000) and generally spreads rapidly through a flock and from flock to flock on the same or neighboring farms.

Mechanical transmission of viruses may occur by people, equipment, vehicles and insects acting as vectors. Domestic flies and darkling beetles have been shown to play a major role in the vectoring of the disease agent (Despins et al. 1994, Calibeo-Hayes et al.

2003). No evidence for a carrier state exists in birds. Wild birds, rodents, and dogs also may serve as mechanical vectors. It was reported that once a farm had been infected with enteric disease, it becomes almost impossible to avert reoccurrence even with extraordinary cleaning and disinfection between flocks (Doerfler et al. 2007).

Enteric pathogens are not transmitted vertically through the egg (Doerfler et al.

2007), but detection of rotaviruses in 3-day-old turkey poults drove speculation that transmission occurs either in or on the egg surface (Theil et al. 1987). It is probable that poults may become infected in the hatchery via contaminated personnel and fomites such as egg boxes from infected farms.

1.4 Diagnostic methods for detection of enteric viruses

Laboratory diagnosis of enteric viruses has been achieved based on virus isolation, electron microscopy, serology, and detection of viral antigens or viral RNA in intestinal tissues or intestinal contents.

1.4.1 Virus isolation. Enteric viruses such as TCoV and astrovirus have been propagated by inoculation of embryonated chicken or turkey eggs with suspensions of intestinal contents, dropping samples, or tissues from suspect infected turkeys (Nagaraja et al.

1997). Virus isolation in embryonated eggs is infrequently applied for the diagnosis of other enteric viruses such as rotaviruses as they are difficult to cultivate in the laboratory

(Devitt et al. 1993). Rotavirus group A infection has been diagnosed by virus isolation in cell cultures, whereas isolation of other rotavirus serogroups in cell culture has proven extremely difficult (Kang et al. 1986, Devitt et al. 1993). As infections with other rotavirus serogroups comprise the majority of rotavirus infections in turkeys (Reynolds et al. 1987), virus isolation in cell cultures may not be the appropriate diagnostic technique.

Another disadvantage of cell culture is the length of time required for the viral induced

23 changes to appear. It may take several virus passages for cytopathic effects to be observed, which usually takes a few days to a few weeks. Also, cell cultures are very susceptible to bacterial contamination and require experienced personnel to perform.

1.4.2 Transmission Electron Microscopy (TEM). The classic way to diagnose enteric virus infections in the laboratory is to identify the virus in feces or intestinal contents by direct electron microscopy. Diagnosis based on electron microscopy requires the identification of virus particles having typical morphology. However, identification of some viruses such as coronaviruses must be distinguished from cell membrane fragments that may resemble coronavirus particles. In addition, diagnosis of some astrovirus may not be accurate with TEM since only a small percentage of viruses may display the characteristic star-shaped morphology. Also, coupled with loss of surface identifying projections, the viruses may be too spaced apart on the grids or among debris hindering positive identification. Because of the relatively insensitive lower detection limit of direct

TEM (106-107 virus particle per milliliter), definitive identification of enteric viruses may be accomplished by immune electron microscopy (IEM). This is achieved by addition of virus-specific antisera to aggregate virus particles thereby aiding virus identification under the electron microscope (Saif et al. 1985). The disadvantage of using TEM is the availability of microscope and staff to operate the equipment. However, TEM or IEM is still valuable in the identification of some uncultivable enteric viruses that may not be isolated by conventional systems in the laboratory.

1.4.3 Polymerase Chain Reaction (PCR) and Reverse Transcriptase - Polymerase

Chain Reaction (RT-PCR). Rapid detection of enteric viruses has important economic and clinical implications. Several PCR and RT-PCR have been developed for detection of enteric viruses including rotaviruses, astrovirus, reovirus, coronavirus and parvovirus in fecal droppings and intestinal contents (Breslin et al. 2000, Tang et al. 2005, Zsak et al.

2009). These PCR assays have been shown to be highly sensitive and highly specific compared to virus isolation, and are valuable for rapid detection of enteric viruses. This procedure is completed by pooling the feces or lower intestines from 3 - 5 birds per flock and either isolating RNA directly or passaging filtered fluids one time through embryonated turkey eggs. The extracted RNA from the field samples or infected embryo intestines then is subjected to PCR using oligonucleotide primers specific for amplification of the viral gene. Multiplex PCR procedures have been described that allow simultaneous detection of some enteric viruses (Spackman et al. 2005a). Such multiplex assay exists for detection of TAstV and TCoV (Sellers et al. 2004a), and also a combination of reovirus, rotavirus and astrovirus was also detected using another multiplex assay (Jindal et al. 2012). The multiplex test can be completed rapidly and provides significant savings in time and materials compared to individual PCR tests.

However, competition for PCR reagents between two or more different targets can affect multiplexing, also stronger targets can be preferentially amplified to the detriment of a weaker target. Also annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction and amplicon sizes should be different enough to identify distinct bands in electrophoresis.

1.4.4 Immunohistochemistry Staining (IHS). Antigen in fresh, frozen or fixed cell suspensions or whole tissues can be detected by the use of specific antibodies labeled with dyes or enzymes in a detection process known as IHS. IHS is valuable in pathogenesis studies as it allows for direct morphologic localization of infectious agents, and can be correlated with corresponding pathologic changes in tissues provided tissue collection is done at the appropriate time during infection. There are different forms of

IHS staining; direct immunofluorescent antibody staining requires labeling the primary antigen-specific antibody with fluorescein dye or an enzyme followed by incubation with the tissue sample. Indirect immunofluorescent antibody (IFA) staining is often used more than direct staining method, and requires incubation of an unlabeled antigen-specific antibody with the tissue sample followed by application of the secondary antibody labeled with fluorescein dye, an enzyme or biotin.

Both direct and indirect fluorescent procedures have been used for detection of enteric virus antigens in intestinal tissues of infected turkeys (Patel et al. 1977). Although direct IHS is simple and economical, it requires conjugation of each primary antibody to a fluorescent dye or an enzyme. This labeling process may be disadvantageous as it may alter the binding capacity of the primary antibody molecule. The sensitivity and specificity of direct FA depends on the quality of the antiserum used to prepare the conjugated antibody, which once produced, may have a relatively short shelf life. Also, the use of direct staining offers little amplification of antigens that are expressed at low levels. Although the indirect method is more complex than the direct staining, it does not

26 require conjugation of primary antibodies, and commercially available flourescein or enzyme conjugated secondary antibodies can be used with numerous primary antibodies for detection. Multiple secondary antibodies can bind to single primary antibody, therefore increasing the sensitivity of this test by enhancing, and intensifying the signal detection.

The sensitivity of indirect IHS is further increased using immunoperoxidase staining (IPS). In addition to the use of fluorescent dyes, other non-fluorescent methods using enzymes such as peroxidase and alkaline phosphatase are now used. These enzymes are capable of catalyzing reactions that give a colored product that is easily detectable by light microscopy. IPS has been successfully applied for the diagnosis of several enteric viral infections and has been described for detection of viral antigens in infected turkey tissues (Breslin et al. 2000, Pantin-Jackwood et al. 2008b, Spackman et al. 2010). Positive staining by IPS is characterized by the presence of intracytoplasmic dark brown granules in infected tissues (Pantin-Jackwood et al. 2008b). Although this method is much more time consuming and expensive than IFA, it provides greater amplification of scarce antigens and provides a more permanent record and histologic detail.

1.4.5 Serology

1.4.5.1 Indirect Fluorescent Antibody (IFA). Detection of specific antibodies to enteric viruses may be accomplished using the indirect fluorescent antibody procedure and have been described for TCoV (Patel et al. 1975). Virus specific antibodies may be detected in

27 experimentally infected turkeys as early as 7 DPI using the IFA (Patel et al. 1975).

Frozen sections of infected embryo intestines or epithelium exfoliated from bursa of

Fabricius of infected turkeys may be used as antigen. Antigen preparation by this method is slow and labor intensive as it requires preparation of frozen sections from intestinal tissues following embryo inoculation which may take up to 72 hours. Nevertheless, this serological method allow discrimination of false positive staining based on determination of the site of specific intestinal staining. However, fluorescent antibody assays are not practical for testing large number of samples and sensitivity is also low compared to RT-

PCR and virus isolation (Breslin et al. 2000).

1.4.5.2 Enzyme Linked Immunosorbent Assay (ELISA). ELISA is a diagnostic method for quantitatively determining antibody concentrations from serum in a multi- well plate format usually 96-well plate. Briefly, antigens in solution are adsorbed to

ELISA plates and antibodies specific for the antigen of interest are used to probe the plate. Background is minimized by optimizing blocking and washing method, and specificity is ensured via the presence of positive and negative controls. Detection methods are usually colorimetric or chemiluminescence based.

The inability to adapt enteric viruses such as TCoV to grow in cell culture has hindered the development ELISA assays for the detection of antibodies as there was no convenient source of antigens. Therefore, other commercially available antigens have been used for detection of antibodies for enteric virus which include the use of IBV whole virus antigen in an antibody capture ELISA (Gomaa et al. 2009). However, the

28 availability of the full length genome sequence of TCoV enabled the production of recombinant antigens for the development of diagnostic tests. Recombinant TCoV-N protein derived from a prokaryotic expression system has been used in antibody ELISAs

(Gomaa et al. 2008) and N-protein produced in a baculovirus expression system has been used in a competitive ELISA (Guy et al. 2002). Furthermore recombinant spike protein antigen produced in E coli has also been used as antigen in ELISA (Gomaa et al. 2009).

The use of recombinant antigens in detection of antibodies has greatly increased the sensitivity and specificity for antibody detection and has been useful for determination of seroprevalence in field serum samples.

1.5 Epidemiology of enteric viruses

The distribution of enteric viruses in turkey flocks appears to be age associated; several longitudinal studies on prevalence have shown that young birds are frequently infected with a variety of enteric viruses. In one study, enteric viruses were present in turkey breeder poults either alone or in combination for up to 9 weeks of age (Jindal et al. 2010).

In another study, the viruses were detected up to 12 weeks of age which was the last sampling period (Pantin-Jackwood et al. 2007). Although there is no prevalence data in birds older than 12 weeks of age, the persistence of virus through feces may maintain infection for an extended period of time (Barnes et al. 2000).

Occurrence of PEC appears to be influenced by environmental conditions and farm location, and typically, the disease tends to become evident during the warmer seasons of the year (Barnes et al. 2000). The severe forms of PEC observed in the

29 southeastern U.S. were reported between May and September, while sporadic outbreaks were reported at other times of the year (Doerfler et al. 2007). This pattern may be attributed to more rapid pathogen multiplication at higher temperatures, resulting to increased environmental contamination, and also to increased numbers and activity of vectors, and reduced resistance of the turkeys as a result of heat stress In time past, outbreaks of coronaviral enteritis in North Carolina have occurred from October through to December, with dramatic increase in enteric virus infections observed following hurricanes Floyd and Irene in eastern North Carolina in the last quarter of 1999. Normal biosecurity and movement of vehicles among farms was probably disrupted by severe flooding and also ease of micro-organism spread was facilitated by the poor environmental conditions (Barnes et al. 2000).

While milder forms of PEC appear to occur throughout the year, its incidence is highest in areas of intense turkey production where farms are situated in less than a mile from each other, particularly involving more than one production company. Although the reasons for this are uncertain, it may be attributed to a high level of pathogen contamination in the environment, the ease of spread of infectious agents among farms in close proximity. The existence of multiple types of enteric pathogens and opportunistic infection, and the density and movement of vectors, and ease of access by infectious agents to susceptible populations are also possible explanations (Barnes et al. 2000).

1.6 Prevention and control

The ubiquity of enteric virus infections in turkeys indicates that it may be impractical to keep commercial flocks free from infection. Presently, no specific treatment or means of control exists. As no vaccines, chemotherapeutics, or other measures are reported to be efficacious for control of enteric infections, the best method to control PEC is to prevent transmission. Good management practices geared at improving cleaning, disinfection and biosecurity particularly, limiting movement of people from farm to farm and resting of facilities between flocks are recommended (Ford et al. 1995). Enteric viruses such as

TCoV can be eliminated from contaminated premises by depopulation followed by thorough cleaning and disinfection of houses and equipment (Patel et al. 1977).

Since rodents can serve as vectors, a strict rodent control program must be implemented to prevent direct contact between wild birds and turkeys. Barns should also be bird-proof, and nesting areas for wild birds eliminated in the environs of the farm.

Since flies and darkling beetles have been implicated as mechanical carriers that can transmit organisms from one flock to another, control programs against these insects area are important. Keeping curtains raised and using power ventilation has been found to be effective in reducing flies in poultry houses (Barnes et al. 2000). Dead turkeys are a reservoir for disease which insects can perch on and it is therefore necessary that carcasses be removed from the flock promptly and buried, incinerated, composted, or disposed of in an approved manner.

The effect of diarrhea on the litter can be minimized by increasing ventilation rate and temperature and by adding fresh litter. Where litter is reused several times, infection

31 will build up, and problems are likely to be more severe than in situations in which houses are cleaned and fumigated and fresh litter is used for each batch of birds.

Immediate removal of litter from a contaminated house is not recommended because there is an increased chance of dispersal and spread of pathogens to other nearby flocks during litter removal due to the relatively high number of organisms present in the litter immediately after the flock is removed (Barnes et al. 2000). Consequently, following cleaning and disinfection, premises should be free of birds for at least 3 – 4 weeks (Guy et al. 2013).

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Chapter 2: Age-related prevalence of enteric viruses in healthy and diseased commercial turkey flocks

2.1 Summary

Enteric viruses in variable combinations have been implicated in enteric diseases in young turkeys, but little is known about their impact and prevalence in older birds. A longitudinal prevalence study of enteric viruses was conducted using fecal samples collected from three turkey commercial farms in Ohio from placement to 21 weeks of age. The results were compared with data obtained with clinical samples collected from

North Carolina, Ohio, Pennsylvania, and Virginia. Samples were screened for the presence of astrovirus, avian rotaviruses group A (AvRV-A) and D (AvRV-D), reovirus, and coronavirus by RT-PCR, and adenovirus and parvovirus by PCR. Regardless of health status or age of the birds, the enteric viruses except adenovirus and coronavirus were frequently detected indicating their widespread circulation in the turkey populations. Astrovirus, parvovirus, and rotavirus were the most frequently detected viruses across all age groups. Astrovirus prevalence and detection rate decreased with age while parvovirus maintained high prevalence across all age. The prevalence of different 43 rotavirus groups greatly varied by age and health condition. AvRV-A was predominantly detected in the first week of age while AvRV-D was the predominant type in 4 weeks of age followed by co-circulation of both groups in older birds in healthy flocks. In contrast,

AvRV-D was the predominant type in diseased flocks regardless of the age of birds. All flocks were negative for TCoV.

2.2 Introduction

Enteric diseases in poultry induce a considerable economic loss in the commercial turkey industry resulting from reduced production due to poor feed conversion efficiency (Guy et al. 1997). Poult enteritis complex (PEC) encompasses a variety of enteric disease syndromes in young turkeys (Breslin et al. 2000). These syndromes include coronavirus enteritis, poult malabsorption syndrome, maldigestion syndrome, and spiking mortality of turkeys with common disease manifestation in turkey poults (Barnes et al. 2000, Yu et al.

2000). The etiology of PEC is believed to be multifactorial, and several enteric viruses have been identified with the condition (Nemes et al. 2008). Enteric viruses commonly associated with these syndromes include astrovirus (Guy et al. 2004), rotavirus (Theil et al. 1987), coronavirus (Ismail et al. 2003), parvovirus (Elena et al. 2011), reovirus

(Heggen-Peay et al. 2002), and adenovirus (Hess et al. 1999).

Astrovirus is known to be widespread in turkey population, and has been the most frequently isolated virus from diseased flocks of young birds with high morbidity and low mortality (Yu et al. 2000). Clinical signs of astrovirus infection vary but include diarrhea, nervousness, litter eating, and growth depression (Guy et al. 1998) with diarrhea

44 being the major clinical manifestation in affected birds (McNulty et al. 1997). Turkey coronavirus has been implicated as an etiology of PEC that was an economically important disease of the U.S. poultry industry (Nagaraja et al. 1997, Guy et al, 1997,

Barnes at al. 2000). However, the virus was not always present in field enteric cases

(Jindal et al, 2009). Reoviruses have been associated with enteric diseases; in addition, it has also been implicated as a cause of viral arthritis in turkeys (Rosenberger et al. 1997).

Adenovirus causes hemorrhagic enteritis in turkeys, and affected birds show clinical signs of depression, bloody diarrhea, and sudden death (Guy et al. 1998). Enteric disease have also been attributed to parvovirus-like particles detected in turkey intestinal tracts

(Trampel et al. 1983). Although high prevalence of parvovirus have been demonstrated in turkey flocks experiencing enteric disease, the role of parvovirus in enteric disease has not been clearly established (Zsak et al. 2009, Murgia et al, 2012).

Beside their presence in clinical cases of enteric disease, enteric viruses have also been detected from the intestines and intestinal contents of apparently healthy commercial turkey flocks (Pantin-Jackwood et al. 2007). Among the enteric viruses, rotaviruses was detected prior to bird placement, thus suggesting possible vertical transmission (Pantin-Jackwood et al. 2007). Different serogroups of rotaviruses have also been detected in healthy and diseased poultry flocks based on electropherotyping analysis in the U.S. in the 1980s (Theil et al. 1987). Avian rotavirus group A (AvRV-A) was detected slightly more in healthy rather than in diseased flocks, while avian rotavirus group D (AvRV-D), also referred to as rotavirus-like viruses, were more prevalent in diseased rather than in healthy flocks (Reynolds et al. 1987). However, recent reports

45 from Europe detected AvRV-A in addition to AvRV-D in flocks with diarrhea, growth depression, and stunting (Otto et al. 2012, Domanska-Blicharz et al, 2013). Recent PCR based surveillance studies have targeted the NSP4 gene of rotavirus (Pantin-Jackwood et al. 2007, Jindal et al. 2010) with which the differentiation of serogroups of avian rotaviruses is not available since NSP4 is a highly conserved gene among groups.

Grouping of rotaviruses have been conducted based on genes encoding the capsid protein, VP6 (Schumann et al. 2009).

The objective of our study was to determine the age-associated patterns of enteric virus infection in healthy commercial turkey flocks and also from turkey flocks with enteric diseases. Phylogenetic analysis of rotavirus VP4, VP6, and VP7 genes was also performed to further characterize the AvRVs detected in this study.

2.3 Materials and Methods

Samples. Fecal samples were collected from apparently healthy turkeys from three flocks in Ohio between October 2011 and March 2012. Flocks belonged to the same farm that was managed by similar operations but employed separate personnel for each farm. Eight fecal samples were randomly collected in each flock at 1, 4, 9, 14, and 19, and from the intestines at slaughter at 21 weeks of age. A total of 161 samples including fecal samples, litter, and intestinal contents were collected between 2000 and 2010 from turkey flocks in

Ohio (3 farms), Pennsylvania (11 farms), Virginia (14 farms), and North Carolina (14 farms). The turkey flocks in Ohio and Pennsylvania were composed of 2- to 7-week-old birds with symptoms of enteric disease including enteritis, stunting, and high mortality,

46 while turkey flocks from Virginia and North Carolina were composed of 2- to 19-week- old birds of which clinical history was unavailable (Murgia et al. 2012) (Table 2.1).

Samples were preserved at -70°C until processed.

Nucleic acid extraction. The fecal samples from healthy and diseased flocks were diluted 1:5 (w:v) in peptone saline diluent and phosphate-buffered saline (pH 7.4), respectively, and centrifuged at 2,000 g for 10 min at 4°C. Fifty microliters of the 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. RNA was also extracted from known turkey rotavirus, astrovirus, coronavirus, reovirus, adenovirus and parvovirus as positive controls. Extracted RNA was subjected to PCR and RT-PCR for the detection of seven viruses as described below.

RT-PCR for astrovirus, coronavirus, rotavirus and reovirus. The RT-PCR conditions and primers for the polymerase gene of astrovirus (Tang et al. 2005), the 3’ untranslated region (UTR) of turkey coronavirus (Culver et al. 2008), the NSP4 gene of rotavirus

(Pantin-Jackwood et al. 2007a), and the S4 gene of reovirus (Pantin-Jackwood et al.

2007b) were used as previously described. The PCR products of 601-bp for astrovirus,

250-bp for coronavirus, 630-bp for rotavirus, and 1120-bp for reovirus were visualized on

1.5% agarose gel.

RT-PCR for detection of avian rotavirus groups A and D. Primers targeting the VP6 gene of AvRV-A, forward primers (5’-ATTCATTTACTCTAACTAGGTCTCA-3’) and the reverse primer (5’-CTGCTACTCCAGGCGTCATAT-3’), were developed to amplify

410-bp fragment with the following conditions: RT at 50°C for 30 min, Taq activation at

94°C for 15 min followed by 35 cycles of denaturation at 94°C for 45 sec, annealing at

51°C for 1 min, and extension at 72°C for 1:30 min, and a final step of extension at 72°C for 10 min. AvRV-D VP6 gene (427-bp fragment) was amplified using forward primer

(5’-TCTAACAATAAATATGTTTTCCT-3’) and reverse primer (5’-

CTAATGTCATCTCTAAATGTCG-3’) with the same conditions except the annealing temperature of 44°C for 1 min. The VP4 and VP7 genes of selected AvRV-A and -D positive samples were also amplified using specific primers as previously described

(Schumann et al. 2009).

PCR for the detection of adenoviruses and parvoviruses. The PCR for adenovirus was performed using published primers targeting 800-bp of hexon gene, HexF1 and HeXR1

(Hess et al. 1999). Parvovirus PCR was performed with primers targeting 561-bp of NS gene of the turkey parvovirus (Zsak et al. 2009). The same PCR conditions were used for the detection of both parvovirus and adenovirus: 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.

Statistical analysis. Prevalence comparisons of each virus among ages, years and states, were analyzed using Kruskal Wallis test, and the prevalence difference between AvRV-A 48 and –D was compared using Mann-Whitney U test (IBM SPSS Statistics 20). A p < 0.05 was considered statistically significant.

Sequencing. Selected rotavirus amplified products from healthy and diseased turkey flocks from different ages were gel purified with QIAquick gel extraction kit (Qiagen,

Valencia, CA) following the manufacturer’s instructions. The purified amplicons were sequenced using forward and reverse primers used for amplification for the VP4 (n=20), and VP6 (n=8) and VP7 (n=23) genes of AvRV-A and -D. The newly determined sequences together with the previously reported sequences available in GenBank were analyzed with the Megalign program using the Clustal W alignment algorithm

(DNASTAR Lasergene, Madison, WI), and a final phylogenetic tree was constructed using Neighbor-Joining algorithm with a bootstrap test of 1,000 replicates.

2.4 Results

A total of 46 turkey flocks from four U.S. states (Ohio, Pennsylvania, Virginia, North

Carolina) were examined by PCR for the prevalence of enteric viruses. The overall presence of enteric viruses in the turkey flocks experiencing enteric problem (diseased), flocks of unknown health condition (unknown), and three turkey flocks with normal performance (healthy) is shown in Table 2.1. The prevalence of parvovirus in diseased and unknown flock samples were previously reported where 71.6% of the samples were positive (Murgia et al., 2012), Overall, over 50% of the flocks examined were positive for astrovirus, parvovirus, rotavirus, and reovirus regardless of age and location, while

49 adenovirus was less frequently detected. No coronavirus was detected in any of the flocks examined.

From three healthy turkey flocks in Ohio, we conducted a longitudinal survey for enteric viruses’ prevalence from 1 week to 21 week of age (Table 2.2). Of the 144 samples, 137 samples tested positive for at least one virus, and 120 samples were positive for multiple viruses. Astrovirus and parvovirus were the most frequently detected viruses

(75.7% and 75.0%, respectively) followed by avian rotavirus which were differentiated into two groups: group A (AvRV-A, 29.2%) and D (AvRV-D, 32.6%). Reovirus was positive in 29.9% of the entire samples, and adenovirus was infrequently detected in a few samples (4.9%).

Similar trends were observed in diseased (Table 2.3) and unknown flocks (Table

2.4) as in the healthy flocks where parvovirus was the most frequently detected viruses followed by rotavirus and astrovirus with a similar detection rate. All the samples from the diseased flock (n=29) were positive for at least one enteric viruses. In the unknown flocks, 134 of 143 samples were positive for one or more viruses and 9 samples were negative. The overall parvovirus detection rate was similar between two groups, but the detection rates of astrovirus, rotavirus, reovirus, adenovirus were higher in the diseased flocks than the unknown flocks: 62.5% and 40.6% for astrovirus, 74.2% and 48.8% for rotavirus, 59.5% and 29.7% for reovirus, and 29.7% and 2.4% for adenovirus in the diseased and unknown flocks, respectively.

Analysis of enteric viruses in different age groups. The virus detection from the samples of healthy, diseased and unknown flocks was compared to analyze any age related difference in astrovirus, parvovirus and reovirus prevalence (Fig 2.1). Since the age distribution varies widely among diseased, unknown, and healthy flocks we arranged the results by age according to that of the healthy flocks: 1 week, 2-4 week, 5-9 week, and so on. Despite the overall high prevalence, the detection rate of astrovirus within a flock tended to decrease from 100% in young birds to 2.7% by 14-week-old in the samples from the unknown flocks and 45.8% by 21-week-old in healthy flocks (p <

0.05). In diseased flocks, which were composed only 2 to 7 weeks of ages, 54% of samples in 2- to 4-week-old birds and 71% in 3- to 7-week-old poults were positive for astrovirus. The parvovirus detection rate in healthy flocks were initially low at 1 week of age (20%) but remained continuously high (over 80%) throughout the survey (p < 0.05).

The parvovirus prevalence in old birds from the diseased and unknown flocks was also similar to our finding from the healthy flocks. Reovirus was frequently but irregularly detected in flocks of different ages. From the samples of diseased and unknown flocks, the detection rate of reovirus was high at 4 and 9 weeks of age, respectively, but it decreased with age, 17.6% in 14-week-old birds from the unknown flocks. In healthy flocks, its detection rates varied widely between 0% and 70.8%.

Group A and D rotaviruses in turkey flocks. Similar to astrovirus and parvovirus, rotavirus was detected with high prevalence in most of the flocks. In healthy flocks

(n=144), we applied group specific PCR to detect and differentiate AvRV-A and AvRV-

D and similar detection rate was observed (29.2% and 32.6%, respectively) (Fig 2.2). In one and four weeks of age, all of three healthy farms were positive for AvRV-A & -D, but the detection rates within flocks were quite different (p < 0.0001) (Table 2.1). We observed the early appearance of AvRV-A in almost 100% of 1-week-old poults (8/8,

8/8, 7/8 from farm 1 to 3, respectively) but few samples were positive for AvRV-D (1/8,

0/8, 0/8). By 4 weeks of age, however, AvRV-D prevailed (8/8, 7/8, 8/8) and only one sample was positive for AvRV-A (1/8, 0/8, 0/8). In diseased and unknown flocks, we initially applied primers targeting NSP4 gene to detect a broad range of avian rotaviruses, which resulted in 74.2% positive samples. All NSP4 positive samples were subjected to another RT-PCR using the AvRV-A & -D specific primers. AvRV-D was predominant in

(p < 0.05) the diseased and unknown flocks (Fig. 2.2). In the diseased and unknown flocks, AvRV-D was present in 68.0% and 23.0% of samples tested while AvRV-A was detected in 4.0% and 1.4%, respectively.

Sequence and phylogenetic analysis of VP6, VP4, and VP7 genes of rotavirus. The partial VP6 gene of AvRV-A (410 bp) and AvRV-D (427 bp) of selected rotaviruses

(n=6) and (n=10) respectively from healthy and disease flocks were sequenced and compared with available sequences of chicken and turkey rotaviruses in GenBank. The nucleotide sequence similarity among AvRV-A or AvRV-D strains from the healthy and diseased flocks in this study were almost identical regardless of age or origin (ranging

99.4% to 100%) while the AvRV-A and -D strains share less than 50% similarity.

Phylogenetic analysis based on the partial VP6 gene sequence further confirmed the close

52 relatedness among viruses from the healthy and diseased flocks for both AvRV-A and

AvRV-D (Fig. 2.3).

The partial VP4 and VP7 gene sequence analyses showed that AvRV-A and

AvRV-D strains from healthy and diseased turkey flocks share nucleotide homology of

89 to 100% in VP4 and 84.2 to 100% in VP7. Phylogenetic tree based on VP4 and VP7 genes showed clustering of AvRVs from this study into multiple groups which may be classified into different VP4 or VP8 seroptypes. However, AvRVs from healthy and diseased flocks did not form separate cluster and randomly clustered with each other or with other previously reported AvRVs (Fig 2.4 and 2.5).

2.5 Discussion

In this study, we examined the prevalence of enteric viruses on the basis of disease status and age of birds. We included a collection of clinical samples from years

2000 and 2005 whose detailed history about the presence of enteric disease were unavailable and treated as random field flocks. Except coronavirus, most enteric viruses examined in this study were frequently present in turkey flocks regardless of the age of birds. Astrovirus, parvovirus, rotavirus, and reovirus were detected with a high prevalence in healthy and diseased turkey. As seen in our longitudinal survey in healthy flocks, the astrovirus detection rate per flock was gradually reduced with age even though it remained high. Also, in diseased and unknown turkeys, the detection rate of astrovirus was reduced with increasing age (Fig 2.1). This age-related trend suggests that older birds

53 are less susceptible to astrovirus and warrants future experimental studies in turkeys of varying ages.

Parvovirus has been identified in turkeys and chickens with enteric diseases (Zsak et al. 2009). In addition to the high prevalence in diseased turkeys (Murgia et al., 2012), parvovirus infections were consistently observed at a high rate across all ages in healthy turkeys. Our results, thus, suggest that parvovirus is broadly distributed in commercial turkeys across different age groups. Parvovirus was detected as early as the first week of age in two of the three flocks. The reason for non-detection of parvovirus from one of the flock is unclear; however, it may be attributed to the small sample size or difference in initial virus contamination at placement.

Although reovirus is often isolated from cases of turkey enteritis as shown in our study, the direct effect of its presence is unclear and experimental studies are lacking. Our study also showed that reovirus prevalence increased with age in healthy turkeys.

Recently reoviruses have been isolated from aged adult turkeys (15- to 18-week-old toms) showing lameness and/or rupture of gastrocnemius tendon and their pathogenesis and relatedness to reoviruses isolated from enteric disease cases is being studied (Mor et al. 2013).

Hemorrhagic enteritis virus (HEV), group 2 adenovirus which was reported in turkeys 4 weeks of age and older (Sharma et al. 1991), was detected in healthy and diseased flocks. Sequence analysis of positive samples from this study revealed identical hexon gene regardless of health status, and the sequences clustered differently from

54 published virulent strains (data not shown). Since no history of HEV was reported in these flocks, the significance of the virus is unknown.

The distribution of rotaviruses in various age groups of healthy turkeys observed in this study is in agreement with a previously observed pattern (Theil et al. 1987), in which only AvRV-A was detected in commercial turkeys less than 1 week of age while other rotavirus-like viruses (RVLV), known as group D rotavirus, were more prevalent in turkeys two-week-old and above. The reason for the predominance of AvRV-A at the first week of age from healthy flocks is unknown. However, a different environmental resistance can be considered between the two groups of AvRV. Rotaviruses have been detected prior to poult placement in the brooder house (Pantin-Jackwood et al. 2007), implying more persistent AvRV-A than AvRV-D in the environment.

Co-circulaton of AvRV-A and -D was observed in two diseased flock samples at

3 and 4 weeks (data not shown) of age and 11% of healthy flock samples. However, a higher prevalence of AvRV-D in diseased turkey flocks was observed in this study as well as in other studies (Reynolds et al. 1987, Theil et al., 1987, Otto et al., 2012,

Bezerra et al., 2012), therefore raising speculations as to its pathogenic effect on the field.

However, the predominance of AvRV-A in diseased flock was also indicated in some studies (Otto et al., 2012, Domanska-Blicharz et al., 2013). In addition, our phylogentic analysis revealed no clear clustering of AvRV-A or AvRV-D based on the VP4 and VP7 genes with regard to age, health status, or year (Fig 2.4 and 2.5).

In conclusion, several enteric viruses except coronavirus were prevalent in turkeys of wide age ranges not only with enteric disease but also in healthy condition.

This study also showed the decreasing trend of astrovirus detection with increasing age and the different distribution of AvRV groups by age and also between diseased and healthy commercial turkey flocks. However, we were not able to relate the specific virus or combination of enteric viruses to manifestation of enteric disease. Systematic survey that include quantitation of the virus combined with experimental challenge studies under different conditions to mimic the different field conditions should be conducted to elucidate the role of these viruses and other factors in the development of enteric disease.

2.6 Acknowledgements

We would like to thank Maria Murgia, Kyung-il Kang, Mohmoud Ibrahim, Mohamed

Elaish and Megan Strother for technical assistance. We also like to thank Dr G.

Rajashekara and Dr Y. M. Saif for provision of field fecal samples. This study was partially supported by the funds appropriated to the Ohio Agricultural Research and

Development Center, The Ohio State University.

2.7 References

Bezerra D. A. et al. (2012). “Detection of avian group D rotavirus using the polymerase chain reaction for VP6 gene.” J. Virol. Methods. 184(2): 189-192. Breslin, J. J., et al. (2000). "Comparison of virus isolation, immunohistochemistry, and reverse transcriptase-polymerase chain reaction procedures for detection of turkey coronavirus." Avian Dis 44(3): 624-631. Culver, F. A., et al. (2008). "RT-PCR detection of avian coronaviruses of galliform birds (chicken, turkey, pheasant) and in a parrot." Methods Mol Biol 454: 35-42. Domanska-Blicharz K. et al. (2013). "Prevalence and molecular characterization of rotavirus from polish turkey flocks between 2008 and 2011." Bull Vet Inst Pulawy 57: 461-464. Elena Alina Palade, Z. D., et al.(2011). "High prevalence of turkey parvovirus in turkey flocks from Hungary experiencing enteric disease syndromes." Avian dis 55: 468-475. Guy, J. S. (1998). "Viral infections of the gastrointestinal tract of poultry." Poultry science 77: 1166-1175. Guy, J. S., et al. (1997). "Antigenic characterization of a turkey coronavirus identified in poult enteritis- and mortality syndrome-affected turkeys." Avian Dis 41(3): 583-590. Guy, J. S., et al. (2004). "Antigenic and genomic characterization of turkey enterovirus- like virus (North Carolina, 1988 isolate): identification of the virus as turkey astrovirus 2." Avian Dis 48(1): 206-211. Heggen-Peay, C. L., et al. (2002). "Isolation of a reovirus from poult enteritis and mortality syndrome and its pathogenicity in turkey poults." 46: 32-47. Hess, M. et al. (1999). PCR for specific detection of haemorrhagic enteritis virus in turkeys, an avian adenovirus, J. Virol. Methods. 81: 199-203. Ismail, M. M., et al. (2003). "Pathogenicity of turkey coronavirus in turkeys and chickens." Avian Dis 47(3): 515-522. Jindal, N., et al. (2010). "Detection and molecular characterization of enteric viruses in breeder turkeys." Avian Pathol 39(1): 53-61. McNulty, M. S., and Guy J. S. (1997). Rotavirus infections. In: Diseases of Poultry.10th ed. B. W. Calnek, H. J. Barnes., C. W Beard, L. R. McDougald, and Y. M. Saif, eds.Iowa State University Press, Ames, IA.: 706-710.

Mor, S. K., et al. (2013). "Isolation and characterization of a turkey arthritis reovirus." Avian Dis 57(1): 97-103.

Murgia, M. V., et al. (2012). "Prevalence of parvoviruses in commercial turkey flocks." Avian Dis 56(4): 744-749. Nagaraja, K. V. and Pomeroy B. S. (1997). Coronaviral enteritis of turkeys (bluecomb disease). In: Diseases of poultry, 10th ed. B. W. Calnek, H. J Barnes., C. W Beard, L. R McDougald, and Y. M Saif, eds. Iowa State University Press, Ames, IA.: 686-692.

Nemes, C., et al. (2008). Astrovirus and rotavirus enteritis in poults in Hungrary., Hungarian. 130: 464-474. Otto, P. H., et al. (2012). "Detection of avian rotaviruses of groups A, D, F and G in diseased chickens and turkeys from Europe and Bangladesh." Vet Microbiol 156(1-2): 8- 15. Pantin-Jackwood, M., J. 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: 235-244. Pantin-Jackwood, M. J., 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., et al. (1987). "A survey of enteric viruses of turkey poults." Avian Dis 31(1): 89-98. Rosenberger, J. K. (2003). Reovirus infection. In: Diseases of poultry, 11th ed. H. J. Barnes. Y.M. Saif, J. R Glisson, A. M. Fadly, L. R. Mcdougal and D. E Swayne (Eds). Iowa State University Press Ames, IA.,: 283-298.

Schumann, T., et al. (2009). "Evidence of interspecies transmission and reassortment among avian group A rotaviruses." Virology 386(2): 334-343. Sharma, J. M. (1991). "Hemorrhagic enteritis of turkeys." Vet Immunol Immunopathol 30(1): 67-71. Tang, Y., et al. (2005). "Development of antigen-capture enzyme-linked immunosorbent assay and RT-PCR for detection of turkey astroviruses." Avian Dis 49(2): 182-188. Theil, K. W. and Saif Y. M. (1987). "Age-related infections with rotavirus, rotaviruslike virus, and atypical rotavirus in turkey flocks." J Clin Microbiol 25(2): 333-337. Trampel, D. W., et al. (1983). "Parvovirus-like enteropathy in Missouri turkeys." Avian Dis 27(1): 49-54. Yu, M., et al. (2000). "Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus." Avian Dis 44(2): 297-304. Zsak, L., et al. (2009). "Development of a polymerase chain reaction procedure for detection of chicken and turkey parvoviruses." Avian Dis 53(1): 83-88. 58

Table 2.1 Summary of the presence of enteric viruses in turkey flocks analyzed in this study. ** Health Age No. Percentage of farms positive State Year condition (week) flock Astrovirus Parvovirus Rotavirus Reovirus Adenovirus Coronavirus Healthy 2011-2012 1-21 3 100 100 100 100 67 0 OH Diseased 2009 2-5 3 100 100* 100 100 67 0 Subtotal 6 100 100* 100 100 67 0

Diseased 2008 7-7 3 67 100* 33 33 67 0 Diseased 2009 3-5 6 67 100* 83 50 17 0 PA Diseased 2010 4-6 3 33 67* 100 100 33 0 Subtotal 12 58 92* 75 58 33 0

1-17, VA Unknown 2000 14 50 93* 86 57 7 0 unknown NC Unknown 2005 3-19 14 57 93* 71 79 0 0

* Parvovirus data in the diseased flocks was included from a previous study (Murgia et al., 2012) ** No. of farms positive/No. of farms tested

Table 2.2 Summary of detected enteric viruses from three different healthy turkey flocks in Ohio from Oct 2011 to March 2012. No. of No. of positive samples Far Age sample AvRV AvRV m (week) AstV Parvo Reo Adeno TCoV s -A -D 1 wk 8 8 0 8 1 0 0 0 4 wk 8 8 8 1 8 0 0 0 OH1 9 wk 8 0 4 0 0 0 5 0 14 wk 8 3 8 0 0 7 0 0 19 wk 8 6 8 0 1 0 0 0 21 wk 8 2 6 0 0 5 1 0 1 wk 8 8 4 8 0 0 0 0 4 wk 8 8 8 0 7 0 0 0 OH2 9 wk 8 8 7 1 0 1 0 0 14 wk 8 8 8 7 8 6 0 0 19 wk 8 5 6 0 0 2 0 0 21 wk 8 6 8 0 6 5 0 0 1 wk 8 8 1 7 0 5 0 0 4 wk 8 8 8 0 8 0 0 0 OH3 9 wk 8 8 7 2 0 1 1 0 14 wk 8 8 6 7 8 4 0 0 19 wk 8 4 5 0 0 2 0 0 21 wk 8 3 6 1 0 5 0 0 Overall detection rate 75.7 75.0 29.2 32.6 29.9 4.9 0.0 (%)

Table 2.3. Summary of enteric viruses from diseased turkey flocks. No. of positive samples Age No. Ade Coro Astr Parv Rota Reo Year State Farm (wee sam novi navi o o* virus virus k) ple rus rus 2009 OH F3 2 5 5 1 5 4 1 0 2009 PA N09-04624 3 1 1 1 1 1 0 0 2009 PA N09-04623 3 1 1 1 0 1 0 0 2009 PA N09-25435 4 1 0 1 1 0 1 0 2009 OH F1 4 8 3 8 5 1 2 0 2010 PA N10-01442 4 1 1 1 1 1 0 0 2010 PA N10-07819 4 1 0 1 1 1 0 0 2009 PA N09-19703 4 1 0 1 1 1 0 0 2009 PA N09-33461 5 1 1 1 1 0 0 0 2009 PA N09-05492 5 1 1 1 1 0 0 0 2009 OH F2 5 4 4 4 2 4 0 0 2008 PA N08-40550 6 1 0 1 0 0 0 0 2010 PA N10-00117 6 1 0 0 1 1 1 0 2008 PA 100138 7 1 1 1 0 0 1 0 2008 PA 100152 7 1 1 1 1 1 1 0 Overall detection rate (%) 62.5 88.0 74.2 59.5 29.7 0.0 * Parvovirus data in the diseased flocks was included from a previous study (Murgia et al., 2012)

Table 2.4. Summary of detected enteric viruses from field samples archived at the Ohio State University during 2000 to 2005 (Murgia et al, 2012). No. of positive samples Sta Age No. Astr Parv Rota Reo Ade Cor Year Farm te (week) samp o o* viru viru novi onav 2000 VA GG #204 1 le1 1 0 0s 0s rus0 irus0 2000 VA PD #1 1 1 1 1 1 0 0 0 2005 NC NC-T-1 3 4 4 0 4 4 0 0 2000 VA SCr #1 3 1 0 1 1 0 0 0 2005 NC NC-T-2 3 12 12 1 8 6 0 0 2005 NC NC-T-3 3 8 8 4 3 5 0 0 2005 NC NC-T-4 3 8 8 3 5 6 0 0 2005 NC NC-T-5 3 7 5 7 2 1 0 0 2005 NC NC-T-6 3 8 8 6 1 3 0 0 2000 VA TB # 9 3 0 3 3 2 0 0 2000 VA TB1059 # 763 11 2 0 1 2 1 0 0 2000 VA BA # 553 12 6 0 5 3 1 0 0 2000 VA CK #344 13 6 0 2 2 1 0 0 2000 VA HA # 13 6 0 2 5 2 0 0 2000 VA HP736 #561-4 14 4 2 4 3 3 0 0 2000 VA DF # 719 14 4 1 3 4 3 0 0 2000 VA GG # 760 17 2 0 1 2 1 0 0 2005 NC NC-T-10 19 8 1 7 0 2 0 0 2005 NC NC-T-11 19 8 0 8 0 1 0 0 2005 NC NC-T-12 19 8 1 6 2 1 0 0 2005 NC NC-T-13 19 9 0 8 1 4 0 0 2005 NC NC-T-14 19 7 0 7 3 1 0 0 2005 NC NC-T-7 19 2 0 2 0 0 0 0 2005 NC NC-T-8 19 2 0 1 0 0 0 0 2005 NC NC-T-9 19 8 0 8 3 0 0 0 2000 VA BD unknown 3 3 3 1 0 0 0 2000 VA MSc unknown 3 2 3 2 0 2 0 2000 VA TK unknown 2 2 2 0 0 0 0 Overall detection rate (%) 40.6 71.3 48.8 29.7 2.4 0.0 * Parvovirus data in the diseased flocks was included from a previous study (Murgia et al., 2012)

Figure 2.1. Prevalence of enteric viruses by age in healthy and diseased flocks and flocks with unknown clinical information. Parvovirus data in the diseased flocks was included from a previous study (Murgia et al., 2012)

100

80 68.0 A D 60

40 29.2 32.6 23.0 (%) rate Detection 20 4.0 1.4 0 N=144 N=25 N=139

Healthy Diseased Unknown

Fig 2.2 Group A and group D avian rotavirus in turkey flocks with different health conditions.

Figure 2.3. Phylogenetic tree using Neighbor-Joining algorithm and representing the genetic relationship of turkey rotaviruses from field samples from healthy (blue) and diseased (red) flocks and previously identified chicken and turkey rotaviruses based on the VP6 gene of rotavirus.

- -

Figure 2.4. Phylogenetic tree using Neighbor-Joining algorithm and representing the genetic relationship of turkey rotaviruses from field samples from healthy (blue) and diseased (red) flocks and previously identified chicken and turkey rotaviruses based on the VP4 gene of rotavirus.

Figure 2.5. Phylogenetic tree using Neighbor-Joining algorithm and representing the genetic relationship of turkey rotaviruses from field samples from healthy (blue) and diseased (red) flocks and previously identified chicken and turkey rotaviruses based on the VP7 gene of rotavirus.

Chapter 3: Effect of Coronavirus Infection on Reproductive Performance of Turkey Hens

3.1 Summary

Turkey coronavirus (TCoV) infection causes enteritis in turkeys of varying ages with high mortality in young birds. In older birds, field evidence indicates possible involvement of TCoV in egg production drops in turkey hens. However, no experimental studies have been conducted to demonstrate TCoV pathogenesis in turkey hens and its effect on reproductive performance. In the present study, we assessed the possible effect of TCoV on the reproductive performance of experimentally infected turkey hens. In two separate trials, 29-30 week-old turkey hens in peak egg production were either mock- infected or inoculated orally with TCoV (Indiana strain). Cloacal swabs and intestinal and reproductive tissues were collected and standard RT-PCR was conducted to detect

TCoV RNA. In the cloacal swabs, TCoV was detected consistently at 3, 5, 7 and 12 days post inoculation (DPI) with higher rates of detection after 5 DPI (>90%). All intestinal samples were also positive for TCoV at 7 DPI, and microscopic lesions consisting of severe enteritis with villous atrophy were observed in the duodenum and jejunum of

TCoV-infected hens. In one of the trials, TCoV was detected from the oviduct of two birds at 7 DPI, however no or mild microscopic lesions were present. In both experimental trials, a 28 – 29% drop in egg production was observed in TCoV infected turkey hens between 4 and 7 DPI. In a separate trial, we also confirmed that TCoV can efficiently transmit from infected to contact control hens. Our results show that TCoV infection can affect the reproductive performance in turkey hens causing transient drop in egg production. This drop in egg production most likely occurred as consequence of the severe enteritis produced by the TCoV. However, the potential replication of TCoV in the oviduct and its effect on pathogenesis should be considered and further investigated.

3.2 Introduction

Coronaviruses are large enveloped, nonsegmented positive sense single stranded RNA viruses belonging to the family Coronaviridae (Cavanagh et al. 2001). Their genome is approximately 30 kb-long from which they transcribe a set of multiple 3’-coterminal nested subgenomic mRNA (Sawicki et al. 2007). The virus possess roughly spherical pleomorphic virions with diameters ranging from 50 to 200 nm with a characteristic petal-shaped spikes on their surface responsible for their crown shaped morphologic appearance as observed using an electron microscope (Dea et al. 1986). The virus particle consists of four major structural proteins including; the highly variable spike (S) glycoprotein, the conserved membrane (M), the nucleocapsid (N), and the small envelop

(E) proteins. The most economically significant coronaviruses affecting the poultry

69 industry belong to the genus Gammacoronaviruses that mainly consist of coronaviruses isolated from birds including turkey coronavirus (TCoV) and infectious bronchitis virus

(IBV) of chicken (de Groot et al. 2008).

TCoV was shown in 1970s to be an important cause of enteric disease of turkeys termed transmissible gastroenteritis, coronaviral enteritis or blue comb disease with severe economic losses to the poultry industry (Nagaraja et al. 1997). The infectious diarrhea caused by TCoV in turkey poults had a negative impact on growth rate and feed conversion efficiency with varying mortality associated with a significant economic loss

(Gomaa et al. 2009). TCoV has also been implicated in severe infectious disease of young turkey poults up to 7 weeks of age termed poult enteritis complex (PEC) with clinical signs including diarrhea, dehydration, stunted growth, inappetence, weight loss, uneven flock growth and dysfunctions of the immune system. When PEC is coupled with clinical manifestation of mortality in young birds, it is also called poult enteritis mortality syndrome (PEMS) (Barnes et al. 2000).

TCoV is antigenically and genetically related to IBV based on studies showing cross reactivity between the two viruses in immunofluorescence and enzyme linked immunosorbent assays (Guy 2000). Also studies showed that the order of the genes at the

3’ ends of the genome of both TCoV and IBV were similar except for the spike glycoprotein gene (Breslin et al. 1999), suggesting that TCoV might have emerged from

IBV (Gomaa et al. 2008). IBV affects chickens of all ages causing highly contagious respiratory disease (Maurel et al. 2011). IBV targets not only the respiratory tract but also the urogenital tract with infection of the oviduct possibly leading to permanent damage in

70 immature birds, and decline in egg production in laying hens (Bisgaard et al. 1976). The production change is also coupled with a decline in the quality of eggs with an increase in number of eggs unacceptable for setting, reduced hatchability, and production of soft shelled, rough shelled and misshapen eggs with loss of shell pigmentation (Bisgaard et al.

1976).

Like IBV, turkey coronavirus has also been linked with rapid drop in egg production and quality in turkey breeder hens (Nagaraja et al. 1997, Clark et al. 2008). It has been speculated that TCoV infection of breeder turkeys usually leads to deterioration in eggshell quality with chalky shells lacking pigmentation, and that the only clinical sign that may appear in breeder turkeys may be a sudden drop in egg production (Clark et al.

2008). Although circumstantial evidence indicates the involvement of TCoV in drops in egg production in laying hens, no experimental studies have been conducted to demonstrate the pathogenesis of TCoV in laying hens. In this study, the potential effect of

TCoV on egg production in layer turkey hens was assessed experimentally for the first time.

3.3 Materials and Methods

Virus preparation. TCoV (Indiana strain) was isolated from intestines of turkey poults with an outbreak of acute enteritis in Indiana. The intestines were homogenized with a 5- fold volume of sterile phosphate-buffered saline (PBS pH 7.2), followed by centrifugation at 2000 g for 10 minutes at 4°C and filtration through 0.45 and 0.22 µm 71 membrane filters (Millipore Products Division, Bedford MA). TCoV NRC-47 was propagated once by inoculation in 22-day-old embryonated turkey eggs via the amniotic route to make a virus stock (Senne et al. 1998). Virus titer was determined using embryonated turkey eggs by inoculating ten-fold serial dilutions of virus. Embryos positive for coronaviral RNA by RT-PCR were recorded and titer was determined using the Reed and Muench method (Thayer et al. 1998).

Experimental infection study to determine the pathogenesis of TCoV in turkey hens.

Two trials were conducted using 26-week-old turkey hens. Turkey hens for trial 1 were obtained from the Ohio Agricultural Research Development Center (OARDC) flock,

Wooster, Ohio, while birds for the second trial were from a commercial farm in Ohio.

The hens were housed in isolation rooms in cages specifically made for laying turkey hens with ad libitum access to feed and water.

Trial 1: Twenty four laying hens were observed until about 90% egg production was attained (approximately the 30th week of age). Birds were separated into 2 groups (n=12 per group); the first group of birds was inoculated orally with 2 ml of the TCoV (~105

EID50/ml) each and the second group with TCoV negative intestinal homogenate derived from PBS inoculated turkey embryos. Egg production was monitored twice daily. At 7 and 14 days post-inoculation (DPI), cloacal swabs were collected from all birds; 4 birds at 7 DPI and the remaining 8 birds at 14 DPI were euthanized and examined for gross pathology and tissues were collected from the intestines (jejunum and ileum) and the reproductive tracts (infundibulum, magnum, isthmus and uterus) in PBS for virus

72 detection. Tissues were also collected for histopathological examination. For this, tissues were fixed by submersion in 10% neutral buffered formalin, routinely processed, and embedded in paraffin. Sections were made at 5 µm and were stained with hematoxylin and eosin (HE). Intestinal contents were also collected from the jejunum and ileum for virus detection.

Trial 2: Forty eight turkey hens were separated into four groups (n=12 birds per group).

At approximately the 29th week of age, groups I and II were inoculated orally with 1 ml

7 of the TCoV preparation (~10 EID50/ml). Group III and IV received TCoV negative intestinal homogenate from PBS inoculated turkey embryos. Egg production was monitored from hens in group I and III twice daily, while birds in groups II and IV were utilized for sample collection. Cloacal swabs were collected from all birds in groups II and IV at 3, 5 and 7 DPI. In addition, 4 birds from groups II and IV, respectively, were euthanized at 7 DPI and intestinal (jejunum and ileum) and reproductive tissues

(infundibulum, magnum, isthmus and uterus) were collected. At the end of the experiment (12 DPI), cloacal swabs, intestinal and reproductive tissues were collected from the remaining birds in groups I and III as in Trial 1.

Experimental infection study to determine the transmission efficiency of TCoV.

Twelve turkey hens from the OARDC flock were used to study the direct transmission of

TCoV from infected to contact control birds. At approximately the 29th weeks of age, 6 birds were inoculated each orally with 1ml of TCoV and housed on the floor of an isolation room with litter. One day after infection, six uninoculated turkey hens (contact

73 control birds) were moved into the same room with the infected birds. Cloacal swabs were collected daily from both infected and contact control birds for 18 days. At 10 and

18 DPI, 2 and 4 birds, respectively, each from infected and contact control birds were euthanized to collect intestinal tissues (jejunum and illeum) for virus detection as described below.

Sample preparation, RNA extraction and RT-PCR. Swab and tissue samples from all experiments were processed as previously described (Chen et al. 2010). Briefly, swab samples were vortexed and then centrifuged at 2000 g for 2 minutes at 4°C to pellet the debris. Tissues were homogenized in sterile PBS (1:5 ratios w/v) and then centrifuged at

2000 g for 10 minutes at 4°C to pellet the tissue debris. RNA was extracted from 200µl of the swab samples and tissue homogenate supernatants using RNeasy Mini kit (Qiagen,

Inc., Valencia, CA) according to the manufacturer’s instruction. All samples collected were subjected to virus detection by standard RT-PCR using a TCoV 3’ untranslated region (UTR) specific primer set to amplify 250-bp as previously described (Culver et al.

2008).

3.4 Results

Pathogenesis of TCoV in turkey hens.

Trial 1. We observed a drop in egg production a day after inoculation which was possibly due to handling stress, in both mock-infected and TCoV infected groups but they returned to normal levels of production by 3 DPI. However, a decline in egg production was observed in the infected bird group between 5 and 7 DPI. During this time, egg 74 production in infected hens declined approximately by 28% as compared with the control group. The egg production in the TCoV infected group returned to a normal level similar to the control group around 8 DPI (Figure 3.1a). Apart from the lowered egg production, no other clinical signs were observed in both experimental groups, neither did we notice any deterioration in egg quality.

Necropsy of infected birds at 7 and 14 DPI revealed severe intestinal pathology.

Intestinal tracts of all birds were inflamed, discolored and distended with yellowish frothy exudates. Microscopically, lesions were similar to reported in TCoV infections of turkey poults (Gomaa et al., 2009; Guy, 2000). Severe enteritis with villous atrophy, with expansion of the lamina propria by inflammatory cells was observed in the duodenum and more consistently in the jejunum at both time points (Figure 3.3a and b).

Sloughing of epithelial cells, congestion and in some cases hemorrhage, and widespread infiltration of lymphocytes and heterophils into the lamina propria of the villi was observed especially at 7 DPI. Also common was the fusion of adjacent shortened villi.

At 12 DPI the inflammatory infiltration of the lamina propria was still pronounced, and numerous enterocytes undergoing division were observed in the crypts. The reproductive tracts also presented with lesions on post mortem examination. Although retention of ovarian follicles was observed in two control birds, the pathological lesions observed in ovaries of TCoV infected turkey hens were much more marked and included retained, pedunculated, ruptured and malformed ovarian follicles. At 7 DPI, two virus inoculated birds had atrophied oviducts with the presence of ruptured yolk in the abdomen that led to egg peritonitis (Figure 3.2a). Microscopically, in the oviduct there was a decrease in

75 number and size of secretory glands in the lamina propria, reflecting their inactivity, but with no signs of inflammation (salpingitis). However, in some hens, loss of the surface epithelium in different sections of the oviduct was also observed (Figure 3.3d). At 14 DPI no microscopic lesions were present in the oviduct of virus infected hens. TCoV was detected in cloacal swabs of all infected birds at 7 DPI by RT-PCR (Figure 3.4) but no virus was detected at 14 DPI. Small and large intestinal contents of all infected hens were also positive for TCoV at 7 DPI, however at 14 DPI, we were able to detect the virus from only one large intestinal sample. The virus was also detected in all parts of the reproductive tract of one bird, and infundibulum and uterus of another hen (Figure 3.4).

We confirmed the TCoV gene by partial sequencing of the 250 base pairs RT-PCR product.

Trial 2. In the second experiment, we separated the groups for monitoring of egg production and sampling to minimize the possible effect of bird handling on the egg production. Similar to the first trial, decline in egg production between 4 and 7 DPI was observed in infected hens (approximately 29%), with egg production returning to normal level at 8 DPI (Figure 3.1b). Consistent with our first trial, no effect on egg quality was observed following experimental inoculation of TCoV. Also, the intestinal pathology observed was similar to that observed in trial 1. The lesions included paleness and congestion of the jejunum and ileum (Figure 3.2b and c), and distention with gaseous frothy contents in the cecum (Figure 3.2d). Microscopic lesions were similar to the observed in trial 1. In contrast to our first trial, the only pathological gross lesion observed in the reproductive tracts of one TCoV inoculated bird was congestion and a 76 prolapsed uterus. No significant microscopic lesions were observed in the oviduct of the

TCoV infected hens. TCoV was detected consistently in fecal swabs from infected birds at 3, 5, 7 and 12 DPI. Half of the cloacal swabs sampled from infected hens at 3 DPI were

TCoV positive, while about 90% of the samples were positive at 5 DPI. TCoV was detected in all cloacal swabs from infected birds collected both at 7 and 12 DPI. The virus was not detected in any part of the reproductive tissues.

Transmission of TCoV to contact control birds. TCoV shedding in fecal swabs of both infected and contact control layer turkey hens is shown in Table 3.1. TCoV was detected as early as 3 DPI from infected birds and 50% of the birds at 5 DPI were positive. The virus shedding increased to about 80% at 6 DPI and all inoculated hens were shedding

TCoV at 7 and 9 DPI. Although the detection rate decreased after 9 DPI, the viral RNA was detected till the end of the experiment (18 DPI). In contact control hens, all swabs were positive for TCoV as early as 3 days post contact (DPC) and about an 80% detection rate was maintained up to 12 DPI. As in inoculated birds, we were able to detect viral RNA till 18 DPI. Consistent with cloacal swab results, all small and large intestinal contents collected from inoculated and contact control hens at 10 DPI (or DPC) were positive for TCoV. Also, 50-100% of the intestinal tissue samples (jejunum and ileum) from inoculated group and contact group, collected at 11 and 18 DPI, and at 10 and 17 DPC, respectively were TCoV positive. Consistent with trials 1 and 2, similar gross pathology lesions were observed in both infected and contact control hens; at 10

77 and 18 DPI, the intestines of birds in both groups were inflamed and distended with gaseous frothy contents respectively.

3.5 Discussion

TCoV is an important viral enteric pathogen of turkeys causing infectious diarrhea (Dea et al. 1986). The disease termed coronaviral enteritis or transmissible gastroenteritis is characterized by decreased weight gain, impaired feed utilization, increased mortality and uneven flock growth in turkeys, and has been incriminated as one of the important causative agents of PEMS (Yu et al. 2000). Turkeys of all ages are affected resulting in increased morbidity and mortality and significant economic loss to the poultry industry

(Ismail et al. 2003, Gomaa et al. 2009). Although TCoV is reported to have a strict tropism for turkey intestinal epithelium and bursa of Fabricius (Guy et al. 2000), field reports indicated TCoV as a potential cause of egg production problems in layer turkeys

(Clark et al. 2008). In addition, using electron microscopy, we detected coronavirus in oviduct of turkey hens that experienced a drop in egg production (unpublished data). In this study, a transient decline, 28 and 29% drop in egg production between 4 and 7 DPI was observed in TCoV infected hens in trial I and II respectively. Also, the peak time points of egg production drop in the first (6 DPI) and second trials (5 DPI), as shown in the boxed area of figure 1 showed about 35% reduction as compared to the control group.

There is therefore no difference between both trials in terms of the transient drop in egg production. Although we detected TCoV in the oviduct tissues of two infected birds at 7

DPI in Trial 1, and mild damage to the oviduct epithelium and glandular depletion was

78 observed, we considered that the transient drop in the egg production may not be due to direct replication of TCoV in the reproductive tracts as the virus detection was not a consistent finding in all samples collected at different time points and/or both experimental trials conducted. On the other hand not every hen had a drop in egg production which might indicate that the decreased egg production occurred only in hens that had severe intestinal infection and pathological changes. No effect on egg quality was observed in our study as opposed to have been reported from field outbreaks (Clark et al. 2008); indicating that other factors in the field, be it environmental, management or concurrent presence of other pathogens may contribute to disease severity as a result of physiological stress. Gross lesions were observed in the ovary of virus infected hens and could explain the drop in egg production; however, microscopic lesions and presence of the virus in the ovaries were not assessed in this study. Future studies examining for the presence of TCoV in reproductive tissues by viral antigen staining

(immunohistochemistry) will help elucidate the role of TCoV in the pathogenesis observed.

The shedding pattern of TCoV in cloacal contents of infected turkey hens was determined by RT-PCR. In our first trial, TCoV could be detected in cloacal swabs at 7

DPI from all inoculated birds but not at 14 DPI. TCoV RNA detection in fecal swabs of infected birds correlated with virus detection in small and large intestinal contents at 7

DPI, in which we found all samples positive, whereas only one weak positive sample was observed at 14 DPI. In the second trial, TCoV was consistently detected in fecal swabs throughout the 12 days of the experimental period. At 3, 5, and 7 DPI, 50%, 90%, and

100%, respectively, of inoculated birds shed the virus. Although TCoV was not detected in fecal swabs at 14 DPI in the first trial, the duration of TCoV shedding in infected layers in our study is almost similar to the shedding pattern observed in poults, in which

TCoV shedding was detected up to 2 weeks after inoculation using the TCoV ATCC R

911 strain (Ismail et al. 2003).

TCoV replicates in the intestines and the virus is shed in feces of infected birds.

Virus shedding in feces may thus be an important avenue for the transmission of TCoV to other birds on the farm, especially for birds housed on the floor with litter. We evaluated the potential of TCoV transmission from infected layer turkey hens to contact hens. The early detection of TCoV shedding in all contact birds at 3 days post contact shows that

TCoV transmits rapidly and efficiently from infected to contact hens via fecal-oral route.

Our findings also suggest that contact hens can shed TCoV to a similar duration as infected hens through re-infection (Table 1). Although our study ended at 18 DPI, based on viral detection from most of the intestinal tissues tested at 18 DPI (Table 1), we expect sporadic detection of TCoV from both infected and contact control hens for a longer period of time.

As an enteric pathogen, TCoV damages intestinal epithelium leading to impaired food absorption, diarrhea and enteritis (Gomaa et al. 2009). In the present study, we observed a progression of intestinal pathology in layer turkey hens challenged with

TCoV. Acute lesions due to the replication of TCoV in intestines of challenged birds were observed at 3 DPI; intestines of inoculated hens appeared thickened and the jejunum and ileum were congested with hemorrhages. These intestinal lesions are consistent with

TCoV replication in poults which has been reported to be primarily in the enterocytes of the jejunum and ileum (Breslin et al. 2000, Guy et al. 2000). We also observed gross pathology in the large intestines. The cecum and colon were markedly distended with gaseous frothy contents. Intestinal distension with gas was more pronounced in birds at 5

DPI, however, by 7 DPI intestinal lesions in infected hens progressed to paleness and thickening of the duodenum and jejunum coupled with markedly distended large intestine. Thickening of the intestines was observed in all twelve infected birds at 12 DPI.

Gross pathology observed in our study was similar to that previously encountered in poults where the intestines of infected poults showed marked enlargement with loose contents (Ismail et al. 2003). The lesions were demonstrated to be associated with depression, anorexia, decreased water consumption, watery diarrhea and poor growth performance (Yu et al. 2000, Ismail et al. 2003).

Gross pathological examination of the reproductive organs also revealed lesions in the ovary and some parts of the oviduct especially in the first trial. At 7 DPI, minimal lesions of retained ova among normal-appearing ovules were observed in control birds; however ovarian lesions were much more pronounced in infected hens with misshapen, mal-developed ovaries; and accumulation of caseous exudates in the peritoneum. The fluid yolk material found in the abdominal cavity of infected birds at 14 DPI may be associated with the rupture of mal-developed ovaries leading to peritoneal adhesions of the oviduct. IBV, a close relative of TCoV has been reported to cause reproductive tract lesions in chickens although severe lesions are observed less in older birds (Alexander et al. 1977).

The decreased egg production in our study is similar to that observed with another enteric pathogen where layer chickens experimentally inoculated with rotavirus showed a drop in egg production from 4-9 DPI (Yason et al. 1987). The egg production decline was attributed to the stress of rotavirus infection. This may also explain the pattern of transient egg production drop observed in our study since egg production decline coincided with severe intestinal pathology and high incidence of virus shedding in fecal swabs. Similar transient decrease in egg production with little or no pathology and virus in the oviduct was also reported in laying chicken hens infected with a low pathogenic avian influenza virus, and in this case it was proposed that the virus infection caused distress or affected feed and water consumption enough to affect lay (Pantin-Jackwood et al. 2012). However, the potential replication of TCoV in the oviduct and their pathogenesis for reproductive tissues should be further investigated. To our knowledge, our experimental study is the first to demonstrate the effect of TCoV infection on reproductive performance of layer turkeys.

3.6 Acknowledgements

We would like to thank Ahmed Ali, Mohmoud Ibrahim and Mohamed Elaish and Megan

Strother for technical assistance. This study was partially supported by the funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State

University.

3.7 References

Alexander, D .J., and Gough R. E. 1977. Isolation of avian infectious bronchitis virus from experimentally infected chickens. Res. Vet. Sci., 23, 344-347.

Barnes, H.J., Guy, J.S., Vaillancourt, J.P., 2000. Poult enteritis complex. Rev Sci Tech 19, 565-588.

Bisgaard, M., 1976. The influence of infectious bronchitis virus on egg production, fertility, hatchability and mortality rate in chickens. Nord Vet Med 28, 368-376.

Breslin, J. J., Smith, L. G., Barnes, H. J., Guy, J. S., 2000. Comparison of virus isolation, immunohistochemistry, and reverse transcriptase-polymerase chain reaction procedures for detection of turkey coronavirus. Avian Dis 44, 624-631.

Breslin, J. J., Smith, L. G., Fuller, F. J., Guy, J. S., 1999. Sequence analysis of the turkey coronavirus nucleocapsid protein gene and 3' untranslated region identifies the virus as a close relative of infectious bronchitis virus. Virus Res 65, 187-193.

Cavanagh, D., Mawditt, K., Sharma, M., Drury, S. E., Ainsworth, H. L., Britton, P., Gough, R. E., 2001. Detection of a coronavirus from turkey poults in Europe genetically related to infectious bronchitis virus of chickens. Avian Pathol 30, 355-368.

Chen, Y. N., Wu, C. C., Bryan, T., Hooper, T., Schrader, D., Lin, T. L., 2010. Specific real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus. J Virol Methods 163, 452-458.

Clark, F. D. 2008. Coronavirus infections in Turkeys. In Avian Advice Newsletter Extension Poultry Veterinarian Center of Excellence for Poultry Science, University of Arkansas Cooperative Extension Service.

Culver, F.A., Britton, P., Cavanagh, D., 2008. RT-PCR detection of avian coronaviruses of galliform birds (chicken, turkey, pheasant) and in a parrot. Methods Mol Biol 454, 35- 42. de Groot, R. J., Ziebuhr, J., Poon, L. L., Woo, P. C., Talbot, P. J., Rottier, P. J., 2008. Taxonomic proposal to the ICTV Executive Committee: revision of the family Coronaviridae. Retrieved 6/22, 2012, from http://talk.ictvonline.org/files/ictv_official_taxonomy_updates_since_the%20_from 8th_report/m/vertebrate-2008/1230/download.aspx.

Dea, S., Marsolais, G., Beaubien, J., Ruppanner, R., 1986. Coronaviruses associated with outbreaks of transmissible enteritis of turkeys in Quebec: hemagglutination properties and cell cultivation. Avian Dis 30, 319-326.

Gomaa, M. H., Barta, J. R., Ojkic, D., Yoo, D., 2008. Complete genomic sequence of turkey coronavirus. Virus Res 135, 237-246.

Gomaa, M. H., Yoo, D., Ojkic, D., Barta, J. R., 2009. Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada. Avian Pathol 38, 279-286.

Guy, J. S., 2000. Turkey coronavirus is more closely related to avian infectious bronchitis virus than to mammalian coronaviruses: a review. Avian Pathol 29, 207-212.

Guy, J. S., Smith, L. G., Breslin, J. J., Vaillancourt, J. P., Barnes, H. J., 2000. High mortality and growth depression experimentally produced in young turkeys by dual infection with enteropathogenic Escherichia coli and turkey coronavirus. Avian Dis 44, 105-113.

Ismail, M. M., Tang, A.Y., Saif, Y. M., 2003. Pathogenicity of turkey coronavirus in turkeys and chickens. Avian Dis 47, 515-522.

Maurel, S., Toquin, D., Briand, F. X., Queguiner, M., Allee, C., Bertin, J., Ravillion, L., Retaux, C., Turblin, V., Morvan, H., Eterradossi, N., 2011. First full-length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses. Avian Pathol 40, 179-189.

Nagaraja, K.V., and Pomeroy, B. S. 1997. Coronaviral enteritis of turkeys (bluecomb disease). In Diseases of Poultry, 10th ed. B.W. Calnek, H. J. Barnes., C. W. Beard, L. R. McDougald and Saif Y. M. (Eds.). Iowa State University Press, Ames. 686-692.

Pantin-Jackwood, M. J., Smith, D. M., Wasilenko, J. L., Spackman, E., 2012. Low pathogenicity avian influenza viruses infect chicken layers by different routes of inoculation. Avian Dis 56, 276-281.

Sawicki, S. G., Sawicki, D. L., Siddell, S. G., 2007. A contemporary view of coronavirus transcription. J Virol 81, 20-29.

Senne, D. A., 1998.Virus propagation in embryonating eggs. In: A laboratory manual for the isolation, identification, and characterization of avian pathogens, 4th ed. D. E. Swayne, J. R. Glisson, J. E. Pearson, W. M. Reed, M. W. Jackwood, and P. Woolcock, eds. American Association of Avian Pathologists, Kennett Square, PA, pp. 235–240.

Thayer, S. G., and Beard, C.W. Serologic procedure 1998. In: A laboratory manual for the isolation, identification, and characterization of avian pathogens, 4th ed. D. E. Swayne, J. R. Glisson, J. E. Pearson, W. M. Reed, M. W. Jackwood, and P. Woolcock, eds. American Association of Avian Pathologists, Kennett Square, PA. pp. 255–266.

Yason, C.V. Summers B. A., chat K. A. 1987. Pathogenesis of rotavirus infection in various age groups of chickens and turkeys: clinical signs and virology. Am J. Vet Res, 48 (6): 977-983.

Yu, M., Ismail, M. M., Qureshi, M. ., Dearth, R. N., Barnes, H. J., Saif, Y. M., 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.

Table 3.1. Virus detection by RT-PCR in cloacal swabs, jejunum and ileum of TCoV infected and contact control turkey hens DPI/ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DPE* Infected Cloacal swab 1/6** 1/6 2/6 2/6 3/6 5/6 6/6 5/6 6/6 4/6 2/4 2/4 1/4 1/4 1/4 1/4 1/4 1/4 Jejunum 1/2 3/4 Ileum 2/2 2/4 Contact Cloacal swab 0/6 0/6 6/6 6/6 6/6 2/6 5/6 5/6 5/6 3/4 3/4 1/4 0/4 0/4 0/4 2/4 1/4 Jejunum 1/2 2/4

86 Ileum 2/2 3/4

*DPI: Days post inoculation for infected hens. DPE: Days post exposure for contact hens **No. of hens positive with RT-PCR/total No. of hens

8619

Control Infected

90 80 70 60 50 40

30 20 10 0 6 DBI 4 DBI 2 DBI 0 DPI 2 DPI 4 DPI 6 DPI 8 DPI

B Control Infected 90 80 70 60

50 40 30 20 10 0 6 DBI 4 DBI 2 DBI 0 DPI 2 DPI 4 DPI 6 DPI 8 DPI 10 DPI

Figure 3.1. Egg production data from infected and uninfected control layer turkey hens in first (A) and second (B) experiments. The X-axis represents days in egg production, 2 days being represented as one unit. The Y-axis represents the percent egg production. Dotted box indicate transient egg production drop between 4-7 dpi. DBI: days before inoculation; DPI: days post inoculation.

A B

j j i

C D

Figure 3.2 Pathological changes produced by coronavirus in experimentally inoculated layer turkey hens. A: ruptured ova at 14 dpi; B: Congested and thickened intestine at 14 dpi (arrow head); C: severely congested jejunum (j) and ileum (i ) mucosa at 3 dpi; D: gaseous and distended cecum (arrow head).

Figure 3.3 Histopathological lesions in laying turkey hens infected with TCoV. A. Jejunum from TCoV-infected turkey hen, 7DPI. Severe enteritis. Sloughing of the epithelium on the tips of the villi (arrow) resulting in blunting and fusion of the villi. Congestion and infiltration of the lamina propria with lymphocytes and some heterophils 200X. B. Duodenum from TCoV-infected hen, 14 dpi. Severe lymphocytic enteritis. 200X. C. Uterus (shell gland) from mock-infected control hens, 7 DPI, 400X. D. Uterus from TCoV-infected hen, 7DPI. Desquamation of the surface epithelium (arrows) and glandular depletion, 400X.

Cloacal swabs Intestinal content

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

SI LI SI LI SI In m Is U In m Is U

Figure 3.4. TCoV detection by RT-PCR in the cloacal swabs, intestinal contents and reproductive tissues of infected layer turkey hens at 7 dpi. Lane M: 100bp DNA marker; Lane 1: TCoV positive control RNA; Lanes 2-5: cloacal swabs; Lanes 6-10: intestinal contents of infected hens (SI: small intestine, LI: large intestine); Lanes 11-18: oviduct compartments (In:infundibulum, m:magnum, Is:isthmus, and U:uterus). Although not shown clearly in the picture, all samples from bird A and infundibulum and uterus from bird B are PCR positives.

Chapter 4: Age-related susceptibility of turkeys to enteric viruses

4.1 Summary

Several different enteric viruses have been identified as causes of gastrointestinal tract infections in poultry with varying morbidity and mortality in young birds. Enteric virus infections are well characterized in poults but limited studies have been conducted in older birds. Susceptibility of 2-, 7-, 12-, 30- and 52-week-old turkeys to turkey coronavirus (TCoV) and turkey astrovirus (TAstV) was evaluated, as well as the effect of combined infection of TAstV and TCoV in 2-week-old poults and turkey hens. From the cloacal swabs and intestines samples, TCoV was consistently detected by PCR throughout the experimental period (1-21 DPI) from all age groups. In contrast, the last detection point of TAstV gradually decreased to 21, 16, and 12 DPI in birds inoculated at

2-, 7-, and 12 weeks of age, respectively, and viral RNA was rarely detected from few samples of cloacal swabs or intestinal contents in turkey hens within 3 DPI. Infection with TAstV alone did not affect body weight in poults or egg production in hens. . The combined infection of TAstV and TCoV did not induce more severe clinical signs and

91 pathology than the TCoV alone infection. However, a severe prolonged decrease in egg production (about 50%) was observed in turkey hens in the co-infection group compared to 20-30% transient drop in coronavirus alone infected hens. The underlying mechanism regarding the age related TAstV susceptibility and the pathogenesis of the TAstV and

TCoV co-infection in layer hens need to be further elucidated.

4.2 Introduction

Enteric diseases impact the turkey industry with substantial economic losses due to decreased weight gain, increased morbidity and mortality, and increased costs of production as a result of poor feed conversion (Guy et al, 1997). Viral enteric disease outbreaks in commercial poultry in recent years have been sporadic with varying degrees of severity (Reynolds et al, 2003), however, the etiologies are complex and multifactorial with several pathogens having similar disease presentation (Pantin-Jackwood et al,

2007a). Poult enteritis complex (PEC) encompasses all infectious intestinal disease of turkey poults between 1 and 4 weeks of age characterized by growth depression, impaired feed conversion efficiency, diarrhea and enteritis (Barnes et al, 2000).

Viruses including turkey coronavirus (TCoV) and turkey astrovirus (TAstV) have been implicated as etiological agents of enteric disease as they have been identified in intestines and fecal contents of diseased turkey flocks (Guy et al, 1997, Barnes et al,

2000, Yu et al, 2000). TAstV is ubiquitous in turkey population and most frequently isolated from diseased flocks primarily between 1 and 4 weeks of age with high morbidity but low mortality (Yu et al, 2000). TCoV has been regarded as the key agent of

PEC in the commercial poultry population (Yu et al, 2000). It was also suggested that

TCoV may affect egg production in layer hens (Nagaraja et al. 1997).

Surveillance studies on both healthy and diseased turkey flocks have revealed the presence of several enteric viruses such as TAstV not only in poults but also in older birds (Jindal et al, 2010, Murgia et al, 2012). Furthermore, various combinations of enteric viruses were observed from healthy commercial flocks (Pantin-Jackwood et al,

2007, Jindal et al, 2010) as well as from flocks suffering enteric diseases (Murgia et al,

2012). Similarly, in our recent survey where we examined the presence of enteric viruses in commercial turkeys up to 21 weeks of age, varying combinations of enteric viruses were detected from apparently healthy flocks (unpublished data). However, their significance and role in the development of enteric diseases in different age groups of turkeys remain unclear and warrant an investigation into the effect of enteric viruses especially in older age groups of birds.

In this study, we investigated the susceptibility of turkeys of different ages to infection with TAstV or TCoV. In addition, we evaluated the effect of combined infection of TAstV and TCoV on weight gain in 2-week-old poults and egg production in hens.

4.3 Materials and Methods

Virus and hyperimmune serum preparations. TCoV (Indiana strain) was isolated from intestines of turkey poults with an outbreak of acute enteritis in Indiana (Yu et al, 2000), and was propagated in 22-day-old embryonated turkey eggs inoculated via the amniotic sac route as described earlier (Senne et al, 1998). The intestines of infected embryos were

93 collected in phosphate-buffered saline (PBS) (pH 7.2) 72 hours post inoculation, homogenized and clarified by centrifugation at 2000 g for 10 minutes at 4°C. The supernatant were filtered through 0.45 membrane filters (Millipore Products Division,

Bedford, MA) and stored at -70°C until use. The TAstV 1987 was obtained from gut content of diarrheic poults (Tang et al, 2006) and working stocks were prepared as described above for TCoV. Virus titer was determined using embryonated turkey eggs by inoculating ten-fold serial dilutions of virus. Embryos positive for TCoV and TAstV

RNA by RT-PCR were recorded and the titer was determined using the Reed and

Muench method (Reed and Muench, 1938).

Hyperimmune antiserum was prepared against the TCoV and TAstV isolates in SPF turkeys using previously published methods (Gelb and Jackwood, 2008). Briefly, 2-

7.0 week-old SPF turkeys were given orally 0.2ml of inoculum containing 10 EID50

7.4 doses/ml of TCoV and 10 EID50/ml of TAstV. Two weeks after initial inoculation, the birds were injected intravenously with the same dose of viruses and serum was collected

2 weeks after intravenous injection. The serum was heat inactivated at 56°C for 30 min and stored at –20°C until used.

Indirect fluorescent antibody (IFA) assay. Intestines from embryo inoculated with

TCoV and TAstV were used as antigen for the detection of serum antibody. Intestines were harvested 4 days after inoculation and embedded in the embedding medium and sectioned at -20°C. Frozen intestinal sections were fixed in cold acetone for 10 min, blocked with 1:10 dilution of Power Block (Biogenex, Fremont, CA) and incubated

94 subsequently with turkey anti-TCoV antiserum at room temperature for an hour. Tissue sections from uninfected embryonated turkey eggs were prepared for comparison as a negative control. After washing with PBS for 10 min, the sections were incubated with

1:100 dilution of fluorescein isothiocyanate (FITC) conjugated goat anti-turkey IgG (H +

L) (Kirkegaard & Perry Laboratories, Gaithersburg, MD) at room temperature in the dark for an hour. Slides were washed in PBS for 10 min, air dried, and mounted on cover glass in Vectshield mounting medium (Vector Laboratories, Burlingame, CA). The slides were examined by a fluorescent microscope.

Susceptibility of different ages of turkeys to TAstV and TCoV infection. The susceptibility of turkeys to TAstV and TCoV was investigated in 2-, 7- and 12-week-old turkeys. Birds were obtained from a flock maintained at the Ohio Agricultural Research

Development Center (OARDC), Wooster, Ohio.

Thirty two 2-week-old poults were separated into four groups (n= 8 per group) in isolation units and weighed. Birds were inoculated by the oral route with 0.2 ml of one of

7.4-7.7 7.0-7.2 the following inoculums: TAstV (10 EID50/ml), TCoV (10 EID50/ml), combination of 0.1ml of TAstV and 0.1ml of TCoV, and intestinal homogenate from PBS inoculated turkey embryos as a control. In study with 7- and 12-week-old turkeys, birds were separated into two groups (n= 6 per group). The birds were orally inoculated with

0.5ml of TAstV and TCoV.

In all groups, blood was collected at 0 DPI and cloacal swabs were collected from all birds at 1, 3, 5, 7, 12, 16 and 21 DPI for virus detection. At 7 DPI, two and four birds/group were euthanized and examined for gross pathology in birds inoculated at 2

95 weeks, and 7 and 12 weeks-old, respectively. Intestines (jejunum and ileum) from 2- week-old birds were collected in PBS for virus detection, and in 10% buffered formalin for histopathological examination. At the end of the experiment (21 DPI), blood and intestines were collected from the remaining birds.

Effect of TAstV and TCoV infection on layer turkey hens. The effect of single TAstV or TCoV infection, and combined infection of TCoV and TAstV on the reproductive performance of turkey hens was investigated at the peak (30 - 32 week-old) of egg production, and also in 52-week-old hens. In all trials, egg production was monitored twice daily. Blood was collected before infection and also at the end of the experiment.

In the first trial, twenty-four hens were observed until about 90% egg production was attained (approximately the 30th week of age). Birds were separated into 3 groups

(n=8 per group); the first group of birds were inoculated orally with 1ml of TAstV (107.4

EID50/ml), and the second group with a combination of 1ml each of TAstV and TCoV

7.0 (10 EID50/ml). Turkey hens in the third group received 1ml of control intestinal homogenate derived from PBS inoculated turkey embryos. Cloacal swabs were collected daily from three birds per group for 21 days. All birds were euthanized and examined for gross pathology.

In the second trial, thirty six 52-week-old turkey hens were separated into three groups (n=12 birds per group) and challenged as in the first trial described above. Cloacal swabs were collected every other day from six birds per group. Four and 2 hens were euthanized at 4 and 7 DPI, respectively, and the remaining birds at the end of the

96 experiment. Birds were examined for gross pathology and intestines (jejunum and ileum) were collected for virus detection.

In the third trial, thirty six 32-week-old turkey hens were separated into four groups. Birds in first two groups (n=12 per group) were orally inoculated with TCoV or a combination of TCoV and TAstV using the same stock of virus with the same titer as in previous trials. The additional twelve hens were separated into two groups (n=6 per group), inoculated with TAstV and TAstV+TCoV combination, respectively, and served for sample collection for virus detection. At 1 and 3 DPI, 3 hens were euthanized from each group and cloacal swabs, intestinal contents and intestinal tissues (duodenum, jejunum, ileum) were collected for virus detection.

Histopathology. Sections of the intestines collected at 7 DPI from 2-week-old poults were evaluated for degeneration, vacuolation, villous atrophy, blunting of villi, crypt epithelial hyperplasia and infiltration of lamina propria with inflammatory cells. Lesions were scored on the basis of severity as follows: nonspecific microscopic, mild, moderate or severe lesions.

Sample preparation, RNA extraction and RT-PCR. Swab and tissue samples from all experiments were processed as previously described (Chen et al. 2010). Briefly, swab samples, which were collected in 2ml PBS, were vortexed and centrifuged at 2000g for

10 minutes at 4°C to pellet the debris. Tissue samples were homogenized in sterile PBS

(1:5 ratios w/v) and then centrifuged at 2000g for 10 minutes at 4°C. RNA was extracted from 100µl of the swab samples and tissue homogenate supernatants using a QIAamp

Viral RNA Kit (Qiagen) and RNeasy Mini Kit (Qiagen), respectively, according to the manufacturer’s instruction. Samples collected from TCoV and TAstV challenged birds were subjected to virus detection by standard RT-PCR using a TCoV 3’ untranslated region (UTR) specific primer (Culver et al. 2008) and TAstV polymerase gene specific primers (Tang et al. 2005), respectively.

Virus isolation (VI). VI was used to confirm the PCR results for TAstV and TCoV detection. The selected cloacal swabs collected from 2-, 7- and 12-week-old birds were used to reisolate the virus in embryonated turkey eggs as previously described (Breslin et al. 2000). Three 21-day-old embryonated turkey eggs per sample were inoculated via the amniotic cavity route with 0.2ml of the selected swab samples. At 4 DPI, embryo intestines were collected in 0.5ml of PBS and the presence of virus was confirmed by standard RT-PCR as described above.

Statistical analysis of body weights. Statistical comparison of body weights among 2- week-old poults was carried out by one-way repeated measures ANOVA (SPSS). A P value of ≤ 0.01 was considered significant.

4.4 Results

Pathogenesis of TAstV and TCoV in different ages of turkeys. Diarrhea with frothy watery droppings was observed in 2-week-old poults inoculated with TCoV alone, and in

TCoV and TAstV co-infection group starting at 2 DPI and lasted for about 4 days. Poults in control group and birds inoculated with TAstV alone remained healthy and no clinical

98 signs were observed. Birds challenged with TAstV alone had similar body weight to that of controls at 7 and 21 DPI (Figure 4.1). However, poults inoculated with TCoV and those in the co-infection group had significantly lower body weight compared to the control birds at both time points (P<0.01). No significant difference in body weight was observed between TCoV alone and co-infection groups. During necropsy at 7 and 21

DPI, the intestine of TCoV challenged poults was filled with yellowish, watery frothy contents. Similar gross lesions were observed in the intestines of poults in the co- infection group. No specific microscopic changes were observed in TAstV inoculated birds compared to controls however, turkeys inoculated with TCoV had mild to moderate microscopic lesions in the small intestines. Mild to moderate villous atrophy was most commonly observed. Vacuolar degeneration and necrosis in the epithelial cells at the villi tips, crypt hyperplasia, and infiltration with mononuclear cells and heterophils in the lamina propria, and focal hemorrhage in the mucosa were occasionally observed. Similar lesions were also seen in the intestines of turkeys of the co-infection group in which villous atrophy with blunted tips and infiltration of heterophils in the lamina propria were the most common histopathological findings. Mild crypt hyperplasia was also present in the small intestine.

In the 7- and 12-week-old birds, no clinical signs were observed in either groups of poults inoculated with TCoV or TAstV. At necropsy, small intestines of all TCoV inoculated birds were distended with gaseous frothy contents at 7 DPI. No obvious gross lesions were observed in the intestine of TAstV infected birds compared to controls.

RT-PCR and virus isolation (VI) from cloacal swabs from 2-, 7- and 12-week-old turkeys. Results of RT-PCR and VI for the detection of TAstV in 2-, 7- and 12- week- old turkeys carried out on selected cloacal swab samples are presented in Table 4.1. The duration of TAstV detection from 2-, 7-, and 12-weeks-old birds decreased with age. RT-

PCR detected TAstV RNA throughout the entire experimental period (21 DPI) in 2- week-old birds but the detection end point decreased to 16 and 12 DPI in 7- and 12-week- old birds, respectively. The detection end point by VI was 16, 16, and 5 DPI in 2-, 7-, and

12-week-old birds, respectively.

In all age groups, TCoV was detected by RT-PCR from infected turkeys throughout the experimental period (Table 4.2). Although TCoV was reisolated in partial numbers of birds at the last tested point (21 & 16 DPI) of 7- and 12-week-old birds, VI was consistently positive until the end of the experiment.

Pathogenesis of TAstV and TCoV in layer turkey hens. In trials 1 & 2 (30- & 52- week-old turkey hens, respectively), turkey hens inoculated with TAstV alone did not show any effect on egg production. In contrast, a severe drop in egg production was observed in hens that received the combination of TCoV and TAstV (Fig. 4.2a and 4.2b).

The egg production began decreasing between 2 to 5 DPI and declined to about 50% less than that of control birds, which did not return to the normal level until the end of the experiment (21 & 11 DPI in 30- & 52-week-old hens, respectively). Other than the lowered egg production in the co-infection group, no other clinical signs were observed and no effect on egg shell quality was observed. At necropsy, gaseous distention of the intestines was observed in all birds in the co-infection group similar to the previously

100 observed lesions in hens inoculated with TCoV alone (Awe et al, 2013). Lesions were also observed in reproductive tract of hens in the co-infection group; at necropsy, retained, pedunculated, and malformed ovarian follicles were observed. No reproductive tract lesion was noticed in TAstV infected hens.

In trial 3 (32-week-old hens), similar to trials 1 & 2, hens co-infected with TAstV and TCoV experienced a severe drop in egg production starting at 4 DPI (Fig. 4.2c). As in trials 1 & 2, the egg production of the co-infected hens did not return to the normal level during the experiment period (10 DPI). On the other hand, the egg production in birds infected with TCoV returned to normal at 8 DPI after a temporal decrease of about

25% decline between 5 and 7 DPI. A transient drop in egg production in TCoV infected hens was also reported in our previous study (Awe et al, 2013) where the hens experienced a temporal decline in egg production between 4 and 7 DPI. At necropsy, gaseous distension of intestines was observed at 4 and 7 DPI in both TCoV alone and co- infection groups. The intestines were also severely distended with gases at 10 DPI.

However, no obvious reproductive tract lesion was noticed.

In all trials, TCoV shedding was consistently detected in cloacal swabs in the co- infected groups (Table 4.3 & 4.4). In contrast, TAstV was not detected at all in trial 1

(30-week-old) in all time points tested (1, 3, 5, 7 DPI). In trial 2 (52-week-old), samples from TAstV infected birds at 1, 3, and 7 DPI were negative for TAstV except a weak positive amplification observed at 1 DPI (Table 4.3). Similar to trial 2, TAstV RNA was detected at 1 DPI with weak intensity in one bird (1/6) in the TAstV infected group in trial 3 but neither of hens in the co-infection group in 3 DPI samples were positive (Table

4.4).

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Increased numbers of TAstV positive samples (1/3 to 3/3) were detected from the intestinal contents of the TAstV and the co-infected hens at both 1 and 3 DPI in trial 3.

TAstV was also detected at 1 DPI in the small intestine of one hen (1/3) in the TAstV alone group.

Antibody detection in turkeys of different ages. From sera collected before challenge

(0 DPI), IFA test for TCoV antibody was negative in all of the age groups (i.e., 2-, 7-, 12-

, 30-, 32-, 52-weeks-old) but became positive at the end of the experiment from birds inoculated with the virus indicating specific seroconversion to the virus. Serum antibodies to TAstV were detected in the sera of all birds of all ages prior to infection

(Figure 4.3). In 2-week-old turkeys, IFA was positive up to 1:200 dilutions before infection (0 DPI). Higher antibody titers (positive at 1:4,000 dilution) were observed in birds at 7-, 12-, 30-, 52-week-old at 0 DPI. At 21 DPI, the serum antibody titer was

1:8,000 in 2-week-old poults, while antibody titer in other age groups did not greatly differ in comparison to titers observed before infection.

4.5 Discussion

In the present study, we evaluated susceptibility of turkeys of various age groups to

TCoV and TAstV. In 2-week-old poults, body weight gain before and after infection was compared and used as a primary determinant of disease severity because it can be easily quantified. Our results are in agreement with a previous report using the same TAstV strain in 2-week-old birds as no difference in body weight was observed compared to controls up to 14 DPI (Tang et al, 2006). Our results however are in contrast with

102 previous studies where diarrhea and growth depression were observed in poults exposed at 1 and 3 days of age (Reynolds et al, 1986, Spackman et al, 2010). These findings indicate that the ability of TAstV to produce clinical signs and impact weight gain in poults decrease as the birds grow older. In addition, other factors such as virulence of the virus strains, number of passages in eggs for preparing virus stocks, titer of the inoculum should be considered in interpreting the differences observed among different studies.

On the other hand, the significantly lower body weight observed in TCoV infected birds is in accordance with a previous report (Ismail et al, 2003). The poor body weight gain in the TCoV inoculated birds may be attributed to the damage caused by the virus to the intestinal mucosa as shown by microscopic lesions, thereby affecting nutrient absorption. It has been previously reported that TAstV may induce more severe signs in birds when co-infected with rotavirus (Reynolds et al, 1987). However, our data indicated that the effect of TAstV in co-infection with TCoV was not prominent implying that the growth depression and pathology observed in the co-infection group was mainly attributed to the effect of TCoV.

In addition to the negative effect of TCoV on weight gain in 2-week-old poults, its pathogenic effect on turkey hens was demonstrated in our previous study (Awe et al,

2013) and similar transient drop in egg production was also observed in this study.

Astrovirus alone had no effect on egg production, however, concomitant infection of the hens with TCoV and TAstV resulted in a severe drop in egg production. This result contrasts with no adverse effect of TAstV in 2-week-old birds. The inoculum used for the study was well characterized and effort was made to ensure it was free from other known specific agents. However, we cannot rule out the presence of other unknown enteric

103 viruses or toxic substances in the TAstV inoculum that may have exacerbated the egg production decline in the turkey hens but not sufficient to cause growth depression in turkey poults.

To better understand the influence of age on viral shedding, we assessed how long

TCoV and TAstV were detectable in different age groups of turkeys using RT-PCR and

VI. In all age groups of turkeys, the end point in TCoV shedding could not be determined and both RT-PCR and VI were positive with swabs collected at the last sampling point.

On the contrary, the decreased detection of TAstV both by RT-PCR and VI were observed with increasing age. The viral shedding pattern observed in birds infected with

TAstV may suggest that the age of turkeys might have an important effect on the extent and duration of TAstV infection. IFA study revealed that anti-TAstV antibodies may be present in any age of turkeys reared in a conventional environment; yet, it may not be surprising since TAstV has been known to be ubiquitous in the turkey populations.

However, the relationship between the presence of these anti-TAstV serum antibodies and their protective role from virus infection is unclear. Sequence variations have been shown in capsid protein among TAstVs circulating in turkeys (Pantin-Jackwood et al,

2007a); it is possible that antibodies detected before infection is against a TAstV strains different from the one used in this study which may explain the replication of TAstV in

2-, 7- and 12-week-old birds in the presence of anti-TAstV antibodies. However, the mechanism of resistance to TAstV infection which was observed in layer hens is unclear.

Turkey hens may have been exposed to many different serotypes of TAstV in the course of their growth including the serotype used in this study, which may explain their resistance to TAstV replication.

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The IFA antibody titer of anti-TAstV antibody increased from 1:200 to 1:8,000 (0 and 21 DPI, respectively) in 2-week-old birds, indicating the presence of newly produced

TAstV serum antibodies. However, the anti-TAstV titers before and after infection did not differ in all other age of birds (7-, 12-, 30-, 52-week-old), despite efficient virus replication and thus sufficient virus sensitization in 7- and 12-week-old birds. It is possible that the antibody titer around or beyond such a high level would be difficult to be differentiated due to the limit of our IFA assay.

In conclusion, a reduced susceptibility to TAstV was observed in turkeys with over 30 weeks of age compared to young turkeys up to 12-week-old of age. In contrast, consistent replication of TCoV and its pathological effect were shown in both young and old turkeys indicating that TCoV can act as a primary pathogen with significant impact on turkey production regardless of the age of the birds. The role and underlying mechanism of TAstV on exacerbating the egg production drop when co-infected with

TCoV need to be further elucidated.

4.6 Acknowledgements

We would like to thank Ahmed Ali, Kyung-il Kang, Mahmoud Ibrahim and Mohamed

Elaish and Megan Strother for technical assistance. This study was partially supported by the funds appropriated to the Ohio Agricultural Research and Development Center, The

Ohio State University.

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4.7 References

Awe, O. O., et al. (2013). "Effect of coronavirus infection on reproductive performance of turkey hens." Avian Dis 57(3): 650-656. Barnes, H. J., et al. (2000). "Poult enteritis complex." Rev Sci Tech 19(2): 565-588. Breslin, J. J., et al. (2000). "Comparison of virus isolation, immunohistochemistry, and reverse transcriptase-polymerase chain reaction procedures for detection of turkey coronavirus." Avian Dis 44(3): 624-631.

Chen, Y. N., et al. (2010). "Specific real-time reverse transcription-polymerase chain reaction for detection and quantitation of turkey coronavirus RNA in tissues and feces from turkeys infected with turkey coronavirus." J Virol Methods 163(2): 452-458.

Culver, F. A., et al. (2008). "RT-PCR detection of avian coronaviruses of galliform birds (chicken, turkey, pheasant) and in a parrot." Methods Mol Biol 454: 35-42. Reynolds D. L., et al (1987). "A Survey of enteric viruses of turkey poults." Avian Dis. 31: 89-98. Gelb J, Jackwood, M.W., (2008). Infectious bronchitis. In: A laboratory manual for the isolation, identification and characterization of avian pathogens, 5th ed. L. Dufour-Zavala, D.E Swayne, J.R Glisson, W.M Reed, M.W. Jackwood, P. Woolcock (Eds.), American Association of Avian Pathologists, Kenneth Square, PA.: 146-149. Gomaa, M. H., et al. (2009). "Infection with a pathogenic turkey coronavirus isolate negatively affects growth performance and intestinal morphology of young turkey poults in Canada." Avian Pathol 38(4): 279-286. Gomaa, M. H., et al. (2009). "Virus shedding and serum antibody responses during experimental turkey coronavirus infections in young turkey poults." Avian Pathol 38(2): 181-186. Guy, J. S., et al. (1997). "Antigenic characterization of a turkey coronavirus identified in poult enteritis- and mortality syndrome-affected turkeys." Avian Dis 41(3): 583-590. Ismail, M. M., et al. (2003). "Pathogenicity of turkey coronavirus in turkeys and chickens." Avian Dis 47(3): 515-522. Jindal, N., et al. (2010). "Detection and molecular characterization of enteric viruses in breeder turkeys." Avian Pathol 39(1): 53-61. Murgia, M. V., et al. (2012). "Prevalence of parvoviruses in commercial turkey flocks." Avian Dis 56(4): 744-749. Nagaraja, K. V., and Pomeroy., B.S. (1997). Coronaviral enteritis of turkeys (bluecomb disease). In B.W. Calnek, C.W. Beard, L.R. McDougald & Y.M. Saif (Eds.). Disease of Poultry, Iowa State University Press.: 686-692. Pantin-Jackwood, M. J., et al. (2007a). "Periodic monitoring of commercial turkeys for enteric viruses indicates continuous presence of astrovirus and rotavirus on the farms." Avian Dis 51(3): 674-680. 106

Pantin-Jackwood, M. J., et al. (2007b). "Enteric viruses detected by molecular methods in commercial chicken and turkey flocks in the United States between 2005 and 2006." Avian Dis. 52: 235-244. Reed L. J, Muench, H. (1938). "A simple method for estimation of fifty percent endpoint." The American Journal of Medical Hygiene 27: 493-497. Reynolds D. L. (2003) Multi-causal enteric diseases. pp. In: Saif Y. M., Barnes H. J., Fadly A. M, Glisson L. K., McDougald L. R.,Swayne D. E. (eds) Diseases of Poultry, 11th ed., Iowa SatePress, Ames, Iowa, pp 1169-1170. Reynolds, D. L. and Saif Y. M. (1986). "Astrovirus: a cause of an enteric disease in turkey poults." Avian Dis 30(4): 728-735. Reynolds, D. L., et al. (1987). "Enteric viral infections of turkey poults: incidence of infection." Avian Dis 31(2): 272-276. Senne, D. A. Virus propagation in embryonating eggs. In: A laboratory manual for the isolation, identification, and characterization of avian pathogens, 4th ed. D. E. Swayne, J. R. Glisson, J. E. Pearson, W. M. Reed, M. W. Jackwood, and P. Woolcock, eds. American Association of Avian Pathologists, Kennett Square, PA, pp. 235–240. 1998.

Spackman, E., et al. (2010). "Astrovirus, reovirus, and rotavirus concomitant infection causes decreased weight gain in broad-breasted white poults." Avian Dis 54(1): 16-21.

Tang, Y., et al. (2006). "Pathogenicity of turkey astroviruses in turkey embryos and poults." Avian Dis 50(4): 526-531.

Tang Y., et al. (2005). "Development of Antigen-capture Enzyme-linked Immunosorbent Assay and RT-PCR for detection of Turkey Astroviruses." Avian Dis 49: 182-188.

Yu, M., et al. (2000). "Viral agents associated with poult enteritis and mortality syndrome: the role of a small round virus and a turkey coronavirus." Avian Dis 44(2): 297-304.

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Table 4.1 Comparison of reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation (VI) for detection of TAstV in cloacal swab from experimentally infected turkeys

Days 2 weeks 7 weeks 12 weeks post infection RT-PCR VI RT-PCR VI RT-PCR VI 1 4/4* NT* 4/4 NT 4/4 NT 3 4/4 NT 4/4 NT 4/4 4/4 5 4/4 NT 4/4 NT 4/4 2/4 7 4/4 NT 4/4 NT 4/4 0/4 12 4/4 NT 4/4 4/4 4/4 NT 16 4/4 4/4 4/4 1/4 0/4 NT 21 4/4 0/4 0/4 NT NT NT *Number of turkeys positive/number tested *NT: not tested

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Table 4.2 Comparison of reverse transcriptase-polymerase chain reaction (RT-PCR) and virus isolation (VI) for detection of TCoV in clocal swabs from experimentally infected turkeys

Days post 2 weeks 7 weeks 12 weeks infection RT-PCR VI RT-PCR VI RT-PCR VI 1 4/4* NT 4/4 NT 4/4 NT 3 4/4 NT 4/4 NT 4/4 NT 5 4/4 NT 4/4 NT 4/4 NT 7 4/4 NT 4/4 NT 4/4 NT 12 4/4 NT 4/4 NT 4/4 4/4 16 4/4 4/4 4/4 4/4 4/4 2/4 21 4/4 4/4 4/4 1/4 NT NT *Number of turkeys positive/number tested *NT: not tested

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Table 4.3 Virus detection in cloacal swabs and intestines of 30 and 52-week-old turkeys in trials 1 and 2 by RT-PCR

Days post Infection

Cloacal swabs Intestines

Group RT-PCR 1 3 5 7 4

TAstV alone TAstV 0/3* 0/3 0/3 0/3 NT* Trial 1 TAstV 0/3 0/3 0/3 0/3 NT (30 wk) Co-infection TCoV 0/3 2/3 3/3 2/3 NT TAstV alone TAstV 0/6 0/6 NT 0/2 0/4 Trial 2 TAstV 0/6 0/6 NT 0/2 0/4 (52 wk) Co-infection TCoV 2/6 4/6 NT 2/2 4/4 *Number of turkeys positive/number tested *NT: not tested

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Table 4.4 Virus detection in cloacal swabs, intestinal contents and tissues of 32-week-old turkeys in trial 3 by RT-PCR

Days post infection

Intestinal Cloacal Intestine contents Group RT-PCR 1 3 1 3 1 3

TAstV alone TAstV 1/6* 0/3 1/3 3/3 1/3 0/3 Trial 3 TAstV 0/6 0/3 2/3 1/3 0/3 0/3 (32wk) Co-infection TCoV 6/6 3/3 NT* NT NT NT *Number of turkeys positive/no. tested *NT: not tested

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Control TAstV TCoV TCoV+TAstV 900 800

700 * * 600 500 400

300 * * Mean body weight (g) weight body Mean 200 100 0 0 7 21 Days post infection

Figure 4.1 Effect of enteric viruses on weight gain in 2-week-old poults. * indicate statistical significance at p<0.01.

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A Control TAstV TAstV+TCoV 100 90 80 70 60 50 40 30 20

B Control TAstV TAstV+TCoV

100 90 80 70 60 50 40 30 20

C TCoV TAstV + TCoV 100 90 80 70

60 50 40 30 20 4DBI 2DBI 0DPI 2DPI 4DPI 6DPI 8DPI 10DPI . Figure 4.2 Effect of TAstV and TCoV on egg production in (a) 30- week-old (b) 52-week-old and (c) 32-week-old turkey hens. The X-axis represents days in egg production, 2 days being represented as one unit. The Y-axis represents percent egg production. DBI = days before inoculation; DPI = days post inoculation

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Figure 4.3 Astrovirus serum antibody detection from different age groups of birds before inoculation showing up to the dilution tested (2-week-old) and the last dilution that was positive for antibody 7-, 12- and 30-week- old turkeys.

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Bibliography

Reference for Chapter 1