ABSTRACT

MACALINTAL, LIZZA MAGSOMBOL. Comparative Pathogenicity Studies on Avian

Reoviruses. (Under the direction of Dr. Frank W. Edens)

Poult enteritis and mortality syndrome (PEMS), a condition with multifactorial

etiology is characterized by an acute, contagious enteric disease of turkey poults between the

ages of 2-4 weeks. The current study was conducted to define the role of PEMS-associated

agents on poult performance. In the first study, the “novel” Cornell virus, defined as the

reovirus ARVCU98, a small round virus (SRV or ARVCU98) and a turkey astrovirus, Ohio

State University isolate (TastOSU), were gavaged orally into the crop of turkey poults.

Reduced body weights and reduced relative weights of the bursa of Fabricius, thymus, and

liver were observed in virus-challenged poults. The reduced body weight gain and tissue

atrophy was exacerbated by the presence of E. coli. In study number two, the possibility of

vertical transmission of reovirus via the egg was tested. In ovo inoculation resulted in

pathogenic and metabolic alterations in broilers challenged in ovo at day 9 of embryonation

with ARVCU98 and the field isolated S1733 (1:100 and 1:500 dilution). In a third study,

hyperimmunization of turkey breeder hens against the ARVCU98 reovirus provided limited

protection to progeny as indicated by decreased weight gain and loss of lymphoid organ

integrity in post hatch ARVCU98-challenged poults. Overall these studies demonstrated that

PEMS-associated astrovirus and reovirus affected poult performance by decreasing body

weight and altering lymphoid organ integrity, and the addition of E. coli further exacerbated

these signs under a controlled environment. Additionally, ARVCU98 reovirus is a turkey

isolate, and the evidence presented herein clearly demonstrated that it can infect broilers and

that vertical transmission via the egg is a strong possibility.

ii

DEDICATION

For: Juan Antonio & Jose Diego

iii

CURRICULUM VITAE

Personal:

Name LIZZA MAGSOMBOL MACALINTAL (Fulbright Fellow)

Home Country Philippines

Birth Date July 2, 1968

Permanent Address 21-5 Camella –Sorrento Panapaan, Bacoor, Cavite, Philippines 4102

Local Address 104C Hidden Springs Rd. Cary, NC 27513

Education:

College University of the Philippines at Los Banos

Degree Doctor of Veterinary Medicine

Professional Affiliation:

Name of Agency Bureau of Animal Industry (On- study-leave) Professional Membership:

Fulbright Asso. - North Carolina Chapter (member)

Fulbright Alumni Asso. (member)

Poultry Science Asso. (student member)

Southern Poultry Science Society (student member)

Philippine Veterinary Medical Asso.

UPLB/UPCVM Alumni Asso.

iv

ACKNOWLEDGEMENT

My sincerest gratitude to my major advisor Dr. Frank W. Edens and to my advisory

committee members, Dr. Simon Shane and Dr. Carm M. Parkhust for all the support and

encouragement during the graduate program. To Rizwana Ali for her assistance and gentle

caring nature and to my first advisor Dr. MA Qureshi, thank you.

To the best ever friends I made during my time in North Carolina State University

(and perhaps in America), Ondulla Foye for being a positive influence in my life and always

believing in my abilities and Renee Plunske for her unselfish nature. I thank you for all the

love and genuine friendship. To Jon and Reverie Molina for all the prayers and camaraderie,

I greatly appreciate it. I would also like to acknowledge Dave and Fidz Fernandez for

bringing me closer to the Lord and whose genuine act of kindness and concern, I will cherish

in my heart. And special mention to Rose Antegro, my original mentor.

My sincerest gratitude is extended to the Fulbright Scholarship Board, for giving me

the opportunity to pursue the Master of Science Degree and to David Hadley, my IIE

coordinator, for all the assistance. To my mother, who will always have a special spot in my

heart and to the unfailing support of my siblings, I cannot thank you enough. And finally, to

Juan Antonio and Jose Diego, my precious angels, who served as my constant inspiration, I

gladly dedicate this work.

v

TABLE OF CONTENTS

LIST OF TABLES.................................................................................................................... ix

LIST OF FIGURES................................................................................................................. xii

LISTOF ABBREVIATIONS ....................................................................................................xv CHAPTER 1

REVIEW OF LITERATURE …………………………………………………………………………..1

VIRAL ENTERIC INFECTIONS IN TURKEYS AND BROILERS………………………… 1 ENTERITIS IN TURKEYS................................................................................................3 POULT ENTERITIS AND MORTALITY SYNDROME.......................................................3 History…………………………………………………………………..…….3 Description of the Disease ...............................................................................3 Disease Transmission.......................................................................................4 Description of PEMS-associated agents.........................................................4 Turkey Coronavirus ........................................................................................5 Small Round Virus (SRV) ...............................................................................5 Turkey Astrovirus............................................................................................6 Reovirus – ARVCU98......................................................................................7 Escherichia coli.................................................................................................8 Detection .......................................................................................................................9 Prevention.....................................................................................................................9 ENTERITIS IN CHICKENS ............................................................................................10 AVIAN REOVIRUS ........................................................................................................10 History.............................................................................................................10 Diseases associated with Reovirus ................................................................10 Virus Description ...........................................................................................11 Mode of Infection ...........................................................................................12 Pathogenesis…………………………………………………………………13 Virus replication.............................................................................................14 Virus Detection...............................................................................................17 Prevention.......................................................................................................18 ORGANS ASSOCIATED WITH THE DIGESTIVE TRACT...............................................19 Liver ................................................................................................................19 ENDOCRINE GLANDS ..................................................................................................22 Pituitary Gland...............................................................................................22 Pancreas ..........................................................................................................23 Thyroid............................................................................................................26 Adrenals ..........................................................................................................30 Current Study.................................................................................................31

vi

CHAPTER 2…………………………………………………………..………………………………33

COMPARATIVE PATHOGENICITY STUDY OF PEMS-ASSOCIATED ASTRO AND REOVIRUSES IN THE PRESENCE OF ABSENCE OF E. COLI IN TURKEY POULTS.........................................33

ABSTRACT ...................................................................................................................33

INTRODUCTION...........................................................................................................34

MATERIALS AND METHODS .......................................................................................35

Animal Welfare ..............................................................................................35

Poults...............................................................................................................35

Experiment 1 ..................................................................................................36

Experiment 2 ..................................................................................................36

Body weights...................................................................................................37

Lymphoid organs ...........................................................................................37

Statistical Analysis .........................................................................................37

RESULTS ......................................................................................................................38

Experiment 1 ..................................................................................................38

Trial 1..............................................................................................................38

Trial 2..............................................................................................................38

Experiment 2 ..................................................................................................39

DISCUSSION .................................................................................................................40

CHAPTER 3...............................................................................................................................54 COMPARATIVE PATHOGENICITY OF PEMS-ASSOCIATED ARVCU98 AND THE FIELD ISOLATED S1733 REOVIRUSES ON BROILERS INOCULATED IN OVO ....................................54

ABSTRACT ...................................................................................................................54

INTRODUCTION...........................................................................................................56

MATERIALS AND METHODS .......................................................................................57

Animal Care ...................................................................................................57

Virus ................................................................................................................57

Embryo inoculation and incubation.............................................................57

Animal Husbandry.........................................................................................58

vii

Body and organ weights ................................................................................58

Feather scoring...............................................................................................58

Serum AST and ALT.....................................................................................58

Plasma Chemistry .........................................................................................59

Reovirus ELISA .............................................................................................59

Immunohistochemistry..................................................................................59

Transmission electron microscopy ...............................................................60

Histopathology................................................................................................60

Statistical Analysis .........................................................................................61

RESULTS ......................................................................................................................61

Hatchability and post inoculation mortality................................................61

Body weights and growth ..............................................................................61

Feed conversion..............................................................................................62

Lymphoid Organ weights..............................................................................62

Feather scoring...............................................................................................63

Gross lesions ...................................................................................................63

Histopathology................................................................................................63

Blood Plasma Chemistry ...............................................................................64

Plasma Glucagon, Insulin and Glucose........................................................64

Insulin-like growth factors ............................................................................64

Cellular integrity............................................................................................64

Immunohistochemistry..................................................................................65

Transmission electron microscopy ...............................................................65

DISCUSSION .................................................................................................................68

CHAPTER 4 ...........................................................................................................................113

EFFICACY OF HYPERIMMUNIZING BREEDER TURKEY HENS TO PROTECT PROGENY AGAINST PEMS INFECTION UPON CHALLENGE.....................................113

ABSTRACT .................................................................................................................114

INTRODUCTION.........................................................................................................115

viii

MATERIALS AND METHODS .....................................................................................116

Animal care...................................................................................................116

Animal husbandry .......................................................................................116

Vaccine production ......................................................................................117

Body weights and Lymphoid organ weights..............................................117

Statistical analysis ........................................................................................117

RESULTS ....................................................................................................................118

In vivo studies...............................................................................................118

DISCUSSION ...............................................................................................................119 SUMMARY AND CONCLUSIONS ............................................................................................131

LIST OF REFERENCES ...........................................................................................................137

ix

LIST OF TABLES

CHAPTER 1

TABLE 1.1 ENTERIC VIRAL INFECTIONS ............................................................................2

CHAPTER 2

TABLE 2.1 THE EFFECT OF SMALL ROUND VIRUS (SRV) AND E. COLI ORAL CHALLENGE ON GROWH OF CONVENTIONAL POULTS (TRIAL 1) .....45

TABLE 2.2 THE EFFECT OF SMALL ROUND VIRUS (SRV) AND E. COLI ORAL CHALLENGE ON GROWH OF CONVENTIONAL POULTS (TRIAL 2) .....46

TABLE 2.3 EFFECT OF SMALL ROUND VIRUS (SRV) AND E. COLI ORAL CHALLENGE ON RELATIVE ORGAN WEIGHT OF CONVENTIONAL POULTS (TRIAL 1)................................................................................................47

TABLE 2.4 THE EFFECT OF SMALL ROUND VIRUS (SRV) AND E. COLI ORAL CHALLENGE ON RELATIVE ORGAN WEIGHT OF CONVENTIONAL POULTS (TRIAL 2)................................................................................................48

TABLE 2.5 THE EFFECT OF ARVCU98, ASTROVIRUS AND E. COLI ORAL CHALLENGE ON BODY WEIGHT (G) ON CONVENTIONAL POULTS....49

TABLE 2.6A RELATIVE ORGAN WEIGHT (%) OF CHALLENGED POULTS AT 4 DAYS POST INOCULATION...............................................................................50

TABLE 2.6B RELATIVE ORGAN WEIGHT (%) OF CHALLENGED POULTS AT 6 DAYS POST INOCULATION...............................................................................51

TABLE 2.6C RELATIVE ORGAN WEIGHT (%) OF CHALLENGED POULTS AT 9 DAYS POST INOCULATION...............................................................................52

TABLE 2.6D RELATIVE ORGAN WEIGHT (%) OF CHALLENGED POULTS AT 11 DAYS POST INOCULATION...............................................................................53

CHAPTER 3

TABLE 3.1. PERCENT HATCHABILITY AND VIABLITY OF FERTILE EGGS INOCULATED WITH REOVIRUS ISOLATED ...............................................76

TABLE 3.2 BODY WEIGHTS OF BROILER CHICKS INOCULATED WITH REOVIUS ISOLATES AT DAY 9 OF EMBRYONATION (TRIAL 1)................................76

x

TABLE 3.3 BODY WEIGHTS OF BROILER CHICKS INOCULATED WITH REOVIRUS ISOLATES AT DAY 9 OF EMBRYONATION (TRIAL 2)..........77

TABLE 3.4 OVERALL MEAN GROWTH INDEX OF BROILERS FROM 1-28 DAYS OF AGE ..........................................................................................................................77

TABLE 3.5 FEED CONVERSION RATIO OF BROILERS INOCULATED IN OVO WITH REOVIRUS ISOLATES AT DAY 9 OF EMBRYONATION ................78

TABLE 3.6A EFFECTS ON RELATIVE LYMPHOID ORGAN WEIGHTS (%) OF BROILERS INOCULATED IN OVO WITH REOVIRUS ISOLATES ON AT 14 DAYS OF AGE (TRIAL 1) .........................................................................79

TABLE 3.6B EFFECTS ON RELATIVE LYMPHOID ORGAN WEIGHTS (%) OF BROILERS INOCULATED IN OVO WITH REOVIRUS ISOLATES ON AT 28 DAYS OF AGE (TRIAL 1) .........................................................................79

TABLE 3.7A EFFECTS ON RELATIVE LYMPHOID ORGAN WEIGHTS (%) OF BROILERS INOCULATED IN OVO WITH REOVIRUS ISOLATES ON AT 14 DAYS OF AGE (TRIAL 2) .........................................................................80

TABLE 3.7B EFFECTS ON RELATIVE LYMPHOID ORGAN WEIGHTS (%) OF BROILERS INOCULATED IN OVO WITH REOVIRUS ISOLATES ON AT 28 DAYS OF AGE (TRIAL 2) .........................................................................80

TABLE 3.8 FEATHER SCORES OF BROILERS CHALLENGED WITH REOVIRUS ISOLATES AT DAY 9 OF EMBRYONATION...................................................81

TABLE 3.9 NECROPSY OBSERVATIONS ON BROILERS CHALLENGED WITH REOVIRUS ISOLATES AT DAY 9 OF EMBRYONATION.............................81

TABLE 3.10A HISTOPATHOLOGY OF THYMUS FROM BROILER CHICKENS TREATED IN OVO WITH REOVIRUS OR PBS, TRIAL 1 .............................82

TABLE 3.10B HISTOPATHOLOGY OF BURSA FROM BROILER CHICKENS TREATED IN OVO WITH REOVIRUS OR PBS, TRIAL 2 .............................82

TABLE 3.11 OVER ALL PLASMA PROFILE OF BROILERS CHICKS CHALLENGED WITH REOVIRUS ISOLATES OR PBS AT DAY 9 OF EMBRYONATION .83

TABLE 3.12 LIVER FUNCTION ASSAY OF BROILER CHICKS CHALENGED WITH REOVIRUS ISOLATES AT DAY 9 OF EMBRYONATION.............................84

xi

CHAPTER 4

TABLE 4.1 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON BODY WEIGHT OF PROGENY AT 6 DAYS AFTER REOVIRUS CHALLENGE..................................................................................121

TABLE 4.2 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS

AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON BODY WEIGHT OF PROGENY AT 10 DAYS AFTER REOVIRUS CHALLENGE..................................................................................122

TABLE 4.3 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS

AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON BURSA WEIGHTS (% of BW) OF PROGENY AT 6 DAYS AFTER REOVIRUS CHALLENGE ....................................................123

TABLE 4.4 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON BURSA WEIGHTS (% of BW) OF PROGENY AT 10 DAYS AFTER REOVIRUS CHALLENGE ....................................................124

TABLE 4.5 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON THYMUS WEIGHTS (% of BW) OF PROGENY AT 6 DAYS AFTER REOVIRUS CHALLENGE ....................................................125

TABLE 4.6 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON THYMUS WEIGHTS (% of BW) OF PROGENY AT 10 DAYS AFTER REOVIRUS CHALLENGE ....................................................126

TABLE 4.7 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS

AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON SPLEEN WEIGHTS (% of BW) OF PROGENY AT 6

DAYS AFTER REOVIRUS CHALLENGE ....................................................127

TABLE 4.8 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON SPLEEN WEIGHTS (% of BW) OF PROGENY AT 10

DAYS AFTER REOVIRUS CHALLENGE.....................................................128

TABLE 4.9 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON LIVER WEIGHTS (% of BW) OF PROGENY AT 6

DAYS AFTER REOVIRUS CHALLENGE.....................................................129

TABLE 4.10 EFFECT OF HYPERIMMUNIZATION OF TURKEY BREEDER HENS AGAINST ARVCU98 REOVIRUS ON POST HATCH ARVCU98 CHALLENGE ON LIVER WEIGHTS (% of BW) OF PROGENY AT 10

DAYS AFTER REOVIRUS CHALENGE ......................................................130

xii

LIST OF FIGURES

CHAPTER 1

REVIEW OF LITERATURE

FIGURE 1.1 REOVIRUS PARTICLE.........................................................................................12

FIGURE 1.2 EM OF CELL APOPTOSIS...................................................................................15

FIGURE 1.3 REOVIRUS REPLICATION.................................................................................16

FIGURE 1.4 METABOLISM OF GLUCAGON ........................................................................25

FIGURE 1.5 METABOLISM OF INSULIN ...............................................................................27

CHAPTER 3

FIGURE 3.1 COMPARISON OF WING FEATHERS OF CONTROL AND S1733 CHALLENGED BROILER CHICKENS .............................................................85

FIGURE 3.2 COMPARISO0N OF WING FEATHERS OF S1733 CHALLENGED BROILER CHICKENS AT FOUR WEEKS ........................................................86

FIGURE 3.3 COMPARISON OF WING FEATHERS OF CONTROL AND S1733 CHALLENGED BROILER CHICKENS AT FOUR WEEKS ..........................87

FIGURE 3.4 HISTOPATHOLOGY OF THYMUS FROM CONTROL BROILERS AT 14 DAYS OF AGE AT 400X...................................................................................88

FIGURE 3.5 HISTOPATHOLOGY OF THYMUS FROM ARVCU98 CHALLENGED BROILERS AT 14 DAYS OF AGE AT 400X.......................................................89

FIGURE 3.6 HISTOPATHOLOGY OF THYMUS FROM S1733 CHALLENGED BROILERS AT 14 DAYS OF AGE AT 400X.......................................................90

FIGURE 3.7 HISTOPATHOLOGY OF THYMUS FROM CONTROL BROILERS AT 28 DAYS OF AGE AT 400X ...........................................................................91

FIGURE 3.8 HISTOPATHOLOGY OF THYMUS FROM ARVCU98 CHALLENGED BROILERS AT 28 DAYS OF AGE AT 400X.......................................................92

xiii

FIGURE 3.9 HISTOPATHOLOGY OF THYMUS FROM S1733 CHALLENGED BROILERS AT 28 DAYS OF AGE AT 400X.......................................................93 FIGURE 3.10 HISTOPATHOLOGY OF BURSA FROM CONTROL BROILERS AT 14 DAYS OF AGE AT 400X...................................................................................94

FIGURE 3.11A HISTOPATHOLOGY OF BURSA FROM ARVCU98 CHALLENGED BROILERS AT 14 DAYS OF AGE AT 400X.......................................................95

FIGURE 3.11B HISTOPATHOLOGY OF BURSA FROM ARVCU98 CHALLENGED BROILERS AT 14 DAYS OF AGE AT 400X.......................................................96

FIGURE 3.12 HISTOPATHOLOGY OF BURSA FROM S1733 CHALLENGED BROILERS AT 14 DAYS OF AGE AT 400X.......................................................97

FIGURE 3.13 HISTOPATHOLOGY OF BURSA FROM CONTROL BROILERS AT 28 DAYS OF AGE AT 400X ...........................................................................98 FIGURE 3.14 HISTOPATHOLOGY OF BURSA FROM ARVCU98 CHALLENGED BROILERS AT 28 DAYS OF AGE AT 400X.......................................................99

FIGURE 3.15 HISTOPATHOLOGY OF BURSA FROM S1733 BROILERS AT 28 DAYS OF AGE..........................................................................................100 FIGURE 3.16 EM OF PANCREAS; CONTROL AT 5K ..........................................................101

FIGURE 3.17 EM OF PANCREAS; ARVCU98 AT 5K ............................................................102

FIGURE 3.18 EM OF PANCREAS; S1733 AT 5K ....................................................................103

FIGURE 3.19 EM OF ANTERIOR PITUITARY; CONTROL AT 5K ...................................104

FIGURE 3.20 EM OF ANTERIOR PITUITARY; ARVCU98 AT 5K .....................................105

FIGURE 3.21 EM OF ANTERIOR PITUITARY; S1733 AT 5K .............................................106

FIGURE 3.22 EM OF THYROID; CONTROL AT 5K .............................................................107

FIGURE 3.23 EM OF THYROID; ARVCU98 AT 5K...............................................................108

FIGURE 3.24 EM OF THYROID; S1733 AT 5K .......................................................................109

FIGURE 3.25 EM OF S1733 VIRUS PARTICLE IN THE LIVER AT 7OK ..........................110

xiv

FIGURE 3.26 EM OF ARVCU98 DETECTED IN THE PANCREAS AT 20K ......................111

FIGURE 3.27 NEGATIVE STAINING OF ARVCU98 AND S1733.........................................112

xv

LIST OF ABBREVIATIONS

ATP Adenosine triphosphate

AST Aspartate aminotransferase

ALT Alanine aminotransferase

AVT Arginine vasotocin

BW Bodyweight

C Cortex

CB Chromophobe

CFU Colony forming unit

CL Chromophil

COR Cisternae of reticulum

CPE Cytopathogenic effect

DAB Days after boost

DPI Days post inoculation

E. coli Escherichia coli

EDTA Ethylenediamine tetra acetate

EID Embryo infective dose

ELISA Enzyme linked immunosorbent assay

F Follicle

FDO Avian reovirus strain

FITC Fluorescein isothiocyanate

HPA Hypothalamo-pituitary-adrenocortical axis

IGF Insulin-like growth factor

IgG Immunoglobulin G

IL-1 Interleukin 1

IL-6 Interleukin 6

IZ Inner zone

LHM Chicken hepatoma cell line

M Mitochondria

MT Mesotocin

N Nucleus

NCARS North Carolina Agricultural Research Services

OCT Optimal cutting temperature

PAGE Polyacrilamide gel electrophoresis

PBS Phosphate buffered saline

PEMS Poult enteritis and mortality syndrome

xvi

PE Pseudostratified epithelium

PP Pancreatic polypeptide

RER Rough endoplasmic reticulum

RNA Ribonucleic acid

RT-PCR Reverse transciptase- polymerase chain reaction

SCZ Subscapular zone

SG Secretory granules

SN Serum neutralization

SRV Small round virus

TastOSU Turkey astrovirus – Ohio State University

TCID Tissue culture infective dose

TCV Turkey coronavirus

TEM Transmission electron microscopy

TG Thyroglobulin

TSH Thyroid stimulating hormone

TNF Tumor necrosis factor

T3 Triiodothyronine

T4 Thyroxine

UC Undifferentiated cells

VN Virus neutralization

ZG Zymogen granules

1

CHAPTER 1

REVIEW OF LITERATURE

Viral Enteric Infections in Turkeys and Broilers

Enteric diseases in turkeys and chickens have been linked to various viral organisms

that are enteropathogens, which are capable of mounting enteric infection or conditions in

turkeys (Reynolds, 1991). In 2000 (Barnes et al., 2000), the term poult enteritis complex

(PEC) was used to describe the syndrome encompassing enteric diseases in the turkey poult.

Enteric diseases in poultry are of prime importance because it can cause large economic

losses in the poultry industry. Reynolds (1991) emphasized that 1) enteric diseases may not

necessarily be caused by only one agent and that detection of virus particle(s) does not

necessarily mean that it is the causative agent, 2) it is likely that pathogenesis of the viral

enteritis is complex one, and 3) the potential for detecting new viral agent(s) is difficult even

if the etiologic virus has been identified or implicated.

Because multiple viruses are associated with enteric diseases (Table 1), this study

dealt mainly with the avian reovirus field isolate (S1733), the poult enteritis and mortality

syndrome (PEMS)-associated reovirus (ARVCU98) and astrovirus (TastOSU), and atypical

Escherichia coli (Types I and II).

Avian reovirus (respiratory enteric orphan virus) infection in commercial poultry

industry has been considered a disease with major economic impact and occurs worldwide.

This includes economic losses due to poor hatchability, mortality, vaccination expenses, cost

of medication, culling and processing related activities. The persistence of reovirus does not

necessarily imply infection since it can be isolated from normal, healthy birds, (Rosenberger

2

and Olson, 1991, Robertson, 1984). Furthermore, depending on the origin, reovirus infection

is not only limited to avian species but to mammalian species as well.

In turkeys, reovirus is one of the organisms implicated in PEMS (Heggen-Peay et al.,

2002) and tenosynovitis (Jones, 2000). Reoviruses are considered to be age-linked and dose

dependent for infection to take place. The occurrence of runting and stunting (malabsorption)

in chickens and turkeys is a major concern of poultry raisers. Affected birds fail to reach

desirable market weight at the end of the grow-out period. In 1997 (Montgomery et al., 1997)

isolated a variety of organisms from intestinal homogenates of infected birds, which

primarily included twelve aerobic, two anaerobic bacteria, IBV (infectious bursal disease

virus), a reovirus, and two bacteriophages.

Table 1.1 Enteric viral infections (from Reynolds, 1991).

Virus Type Species affected associated condition/disease

Adenovirus (group II) Turkey Hemorrhagic enteritis Astrovirus Turkey Turkey viral enteritis Corona virus Turkey Blue comb disease Enter virus Turkey Diarrhea/enteritis Orthoreovirus Turkey Turkey viral enteritis Parvoviruslike virus Turkey Enteropathy,stunting,diarrhea Picornalike virus Turkey Enteric and respiratory disease Reovirus Turkey Malabsorption syndrome Rotavirus Turkey/Chicken Diarrhea and enteritis Calicivirus Chicken Infectious stunting syndrome Coronavirus like particles Chicken Malabsorption syndrome Enterolike virus Chicken Infectious stunting syndrome Parvovirus Chicken Infectious stunting syndrome Reovirus Chicken Malabsorption syndrome Toga virus-like agent Chicken Infectious stunting syndrome

3

Enteritis in Turkeys

Poult Enteritis and mortality Syndrome (PEMS)

History

The PEMS condition emerged as an important disease in the mid-90’s and nearly

destroyed the turkey industry in the Southeastern United States. At the time of its appearance,

there was no known etiology for the disease. It was reported initially in a flock of turkeys in

western North Carolina (Barnes and Guy, 1997). Within two years after its emergence,

USDA-ARS reported that PEMS-losses amounted to more than $35 million (ARS Annual

Report, 2003). Recent surveys have suggested that since 1991, PEMS has cost the turkey

industry approximately $15 million per year or about $0.05 per turkey poult placed and

grown to market age. To date, PEMS is classified as a disease with a multifactorial etiology

owing to the fact that several other organisms were found to be associated with the disease

process (Heggen-Peay, 2002b). Viral agents such as coronavirus (Lin, et al., 2002; Guy and

Barnes, 2000; 1989 Yu et al., 2000), adenovirus (Yu et al., 2000), astrovirus (Yu et al., 1998,

2000; Qureshi et al., 2000; Koci et al., 2000) and reovirus (Schat et al., 1998) were

implicated as well as bacterial agents like; salmonella, camphylobacter, clostridia and E. coli

types I and II (Edens, et al., 1997abc).

Description of the Disease

In the fall of 1991, acute enteritis, thymus and bursal atrophy in commercial turkey

poults from North Carolina was reported as turkey spiking mortality (Brown et al., 1997). It

had a rapid onset with mortality of 1% of the flock per day for 1-5 days in 5-25 days old

4

poults. During its emergence PEMS was turkey spiking mortality (Barnes & Guy 1997), but

the description was later called PEMS due to its less severe clinical signs (Barnes, et al.,

2000). Mortality averaged about 12% of all the poults placed in a brooder house and peaked

around 19 days of age. Feed refusal, vocalization, enteritis, diarrhea, decreased growth, high

mortality and flock unevenness were commonly found in poults affected by PEMS. The

spike in PEMS-attributed mortality was correlated with low feed intake, poor nutrient

absorption, hypothermia and hyopo-phosphatemia (Edens, 1997; Edens et al., 1997abc;

Qureshi et al., 1997). Poults that survived the infection were unable to compensate for the

stunting associated with the disease (Odetallah, 2001) because there was impaired nutrient

absorption and energy utilization (Doerfler et al., 2001ab). Further characterization of PEMS

infection revealed that challenged poults had severe malabsorption (Doerfler et al., 2000a).

Depressed blood levels of insulin, thyroxine (T4) and triiodothyronine (T3) were

characteristic for PEMS infected poults (Doerfler et al., 2000b).

Disease Transmission

PEMS can be readily transmitted through direct contact exposure to seeder poults

(Odetallah et al., 2001; Doerfler et al., 1998; Heggen et al., 1998; Qureshi et al., 1997). Co-

housing normal, healthy poults with suspected PEMS-infected poults resulted in decreased

body weight at 6 days post-exposure (Qureshi et al., 1997). As early as 2 days post-exposure

(Doerfler et al., 1998), huddling and clinical signs of PEMS have been reported. Brown et al.

(1997) used litter from previously infected flocks and reproduced spiking mortality within

five days in poults raised on the litter.

5

Accordingly, infection through oral transmission has also been reported.

Experimental oral inoculations of filtered fecal and intestinal contents as well as thymic

materials from infected poults were shown to induce PEMS-like signs (Heggen-Peay et al.,

2002b; Doerfler et al., 2000ab; Qureshi et al., 1999; Schultz-Cherry et al., 2000). Egg

transmission of PEMS has not been reported but that possibility does exist.

Description of PEMS –Associated Agents

Turkey Coronavirus

Turkey corona virus (TCV) was first isolated and associated with poult enteritis (Lin

et al., 1996). Later PEMS infection was found to be induced by TCV negative fecal material

(Carver et al 2001; Barnes et al., 1997). In fact all the works published by Doerfler, Edens

and their colleagues were the result of studies conducted with TCV negative fecal inocula.

When E. coli was challenged into TCV-positive poults, it took about 60 minutes to clear the

bacteria in the bloodstream (Heggen et al., 1998). PEMS positive, TCV-negative poults had a

lower CD4+/CD8+ ratio compared to the PEMS positive, TCV-positive and control poults at

14 days post infection. TCV-positive poults did not diminish the number of cells in the

mononuclear phagocytic system (MPS) but showed an increased macrophage recruiting

ability (Heggen, et al., 1998). These observations suggest that PEMS caused impaired the

MPS and altered lymphocytic populations.

Small round virus (SRV)

A novel small round virus (SRV) was isolated at the Ohio State Agricultural Research

and Development Center from PEMS-positive, TCV-negative fecal material (Yu et al.,

6

2000). It was reported that when the SRV was given orally to turkey poults (Qureshi et al.,

2000), it induced clinical signs similar to those in poults exhibiting a mild form of PEMS and

was characterized by diarrhea, weight loss, lymphoid organ atrophy, and alteration in the

lymphocyte subpopulation as well as reduction in the lymphoproliferative response to

concanavalin A (ConA). When SRV was given in combination with TCV, mortality was

exacerbated (Yu et al., 2000ab). Later studies (Qureshi et al., 2001) revealed that the virus

capsid of the Ohio SRV had a 100% homology with an astrovirus implicated in PEMS (Koci

et al., 2000). The Ohio State University SRV would later be designated as Tast-OSU

(Qureshi et al., 2001)

Turkey Astrovirus

Astrovirus has been found routinely in poults exhibiting diarrhea and enteritis with

unknown etiology (Reynolds et al., 1986). In turkey hatchlings, astrovirus has been

associated with diarrhea (Thouvenelle et al., 1995). It has been suggested that turkey

astrovirus is one of the many etiologic agents linked with development of PEMS. Qureshi et

al. (2001) reported that Tast-OSU challenge induced defects in macrophage effector

functions, implying that PEMS Tast-OSU can potentially impair the immune responsiveness

of turkeys by reducing macrophage viability and decreasing phagocytosis and

intracytoplasmic killing of E. coli. Another astrovirus, TastV (Schultz-Cherry et al., 2000)

was isolated from PEMS-positive, TCV-negative turkey poults. Astrovirus replication can be

inhibited by cellular nitric oxide production (Koci, et al., 2004).

7

Reovirus – ARVCU98

Fecal filtrates (100nm) from PEMS-positive, TCV-negative infected turkey poults

were studied by Heggen-Peay and co workers (2002a). The ARVCU98 reovirus was a novel

virus isolated and described by Heggen-Peay et al. (2002). Oral challenge with this isolate

caused intestinal and cecal inflammation as well as excessive flatulence (Heggen-Peay et al.,

2002a) similar to signs in poults suffering from spiking mortality (Barnes and Guy, 1997).

Lymphoid organ integrity was compromised as evidenced by atrophy of the bursa (75%),

thymus (99%) and spleen (75%), which was similar to the signs of experimentally induced

PEMS (Qureshi et al., 1997). Heggen-Peay et al. (2002) also reported that ARVCU98

decreased relative weights of bursa and thymus.

Thymus has been shown to be an important target in the PEMS pathogenesis. Poults

challenged with thymus filtrate from PEMS-infected birds manifested diarrhea, growth

depression, mortality, pathology, and immunosuppression similar to poults exposed to the

intestinal filtrate (Schultz-Cherry et al., 2000). Liver weight on the other hand was found to

be decreased as a result of oral inoculation with the ARVCU98 (Heggen-Peay, 2002a). The

ARVCU98 virus can be isolated easily at 3-6 days post infection but thereafter virus

shedding diminishes.

The ARVCU98 virus isolate was found to be susceptible to heat. At 80°C the virus

was completely inactivated, but it was only partially inactivated at 60°C. Through

polyacrilamide gel electrophoresis (PAGE) analysis, it determined that ARVCU98 contains

10 segments of double stranded RNA in which the pattern of migration is not similar to that

of the avian reovirus strain (FDO) and the mammalian Dearing strain (Heggen-Peay et al.,

2002a). Using different cell lines, it was observed that ARVCU98 had cytopathic effects

8

(CPE) on a chicken hepatoma cell line (LMH) and primary turkey liver cells. The CPE

includes syncytia formation, cell death and sloughing off of the adherent infected cells. The

liver appeared to be the targeted site of replication because ARVCU98 did not induce the

same CPE on either the macrophage or B cell lines (Heggen-Peay et al., 2002b). This can

explain liver atrophy in PEMS infected poults (Heggen-Peay et al., 2002b).

PEMS induces atrophy of the primary and secondary lymphoid organs leading to

altered immune response (Qureshi et al., 1997). There is enhancement of interleukin-1 (IL-1)

and IL-6 activity and nitrite production and lowered tumor necrosis factor (TNF) (Heggen et

al., 2000; Qureshi et al., 2001). Up-regulation of different macrophage-produced cytokines

appears to have contributed in the inflammatory process in the intestines leading to diarrhea,

increased mucosal permeability, and nitrite production, which has been linked to inhibition of

virus replication (Heggen et al., 2000; Koci et al., 2004).

Escherichia coli

Edens et al., (1997 a, b) isolated two aggressive and one time considered as atypical

E. coli (BBL: 36570 and 34560 for colony types 1 and 2, respectively; API-20E: 5144572

and 5144512 for colony types 1 and 2, respectively) in PEMS infected poults. When given

orally, these isolates caused mortality, diarrhea, weight depression, and cyclophosphamide

treatment further enhanced the response (Edens, et al., 1997a). Additionally, these E. coli

isolates caused the ileal epithelium cell microvilli and subcellular organelles to be damaged,

which contributed to malabsorption of nutrients associated with PEMS infection (Edens et

al., 1997a). These E. coli isolates were very important in determining that PEMS had a

9

multifactorial etiology, and that E. coli might be the ultimate cause of mortality in poults that

had been compromised by either reovirus or astrovirus infection.

Detection

Aside from monitoring clinicopathological findings, surveillance of on-farm activity

is a useful methods of detecting PEMS. Other methods used to detect agents associated with

PEMS infection are immunoflourescent testing for the presence of viral antigen (Heggen-

Peay et al., 2002a;Qureshi et al., 1999) and demonstration of the virus through isolation and

eventual electron microscopic examination. Recently, Koci et al. (2000) developed an RT-

PCR for detection of PEMS-associated astrovirus.

Prevention

The implementation of strict biosecurity, which includes effective cleaning and

disinfection as forms of prevention, has been considered. Since 1.0% formaldehyde was

found to be effective in vitro to inactivate spiking mortality organisms (Brown et al., 1997),

then it is possible to use this as a cleaning /disinfecting agent. No vaccines have yet been

developed for PEMS, but limited use of some antibiotics can be used to avert possible

bacterial infection that complicated the disease. Nutritional intervention resulted in limited

improvement in flock performance (Doerfler et al., 2000a; Roy et al., 2002). The addition of

electrolytes, glucose and citric acid to drinking water improved the humoral immune

responses of poults with PEMS (El Hadri et al., 2004).

10

Enteritis in Chickens

Avian Reovirus

History

The history of avian reovirus was reviewed by Van der Heide (2000). Briefly,

reovirus was first reported by Olson and co-workers in 1957 as the synovitis-inducing agent

and Kerr and Olson (1964) found this agent to be pathogenic in young broilers chickens.

Hence, it was called the viral arthritis agent after determining that the synovitis-inducing

agent was a virus (Olson et al., 1966). This was similar to the Fahey-Crawley virus (Fahey et

al., 1954) originally isolated in chickens infected with chronic respiratory disease and later

confirmed by Petek et al. (1967). It was not until 1972 that this virus was found to be a

reovirus when it was confirmed through electron microscopic analysis (Walker et al., 1972).

Diseases Associated with Reovirus

Aside from viral arthritis and leg weakness-related problems (Goodwin, et al., 1993;

Robertson and Wilcox, 1986; Rosenberger and Olson, 1991), numerous other disease

conditions such as myocarditis, pericarditis (Rosenberger et al., 1988), hepatitis (Mandelli,

1978), splenitis (Hieronymus et al., 1983), bursal atrophy (Kibenge, 1987), enteric problems

and malabsorption syndrome (Kibenge and Wilcox, 1983), respiratory disease (Fahey and

Crawley, 1954), and immunosuppression (Sharma et al., 1994) have been reported in avian

reovirus-infected chickens. Although, avian reovirus mainly affects chickens and turkeys

(Page et al., 1982), it was also found to infect geese (Palya et al., 2003), pheasants (Mutlu et

al., 1998), and quail (Magee et al., 1993).

11

Virus Description

Reovirus (respiratory enteric orphan virus) belongs to the family Reoviridae genus

Orthoreovirus (Urbano, et al., 1994). Avian reovirus differs from mammalian isolates in that

it lacks hemagglutination activity (Glass et al., 1973), lacks ability to induce cell fusion

(Wilcox, 1982), and is associated with naturally occurring pathological condition (Robertson

and Wilcox, 1986). Additionally, if one is using the virus neutralization test, no cross-

reaction between avian and mammalian viruses has been observed (Spandidos and Graham,

1976). Similarities between avian and mammalian viruses do exist- both are RNA viruses,

they are heat, ether and chloroform resistant, and particle size is between 50-100nm in

diameter (Desmukh and Pomeroy, 1969b). The structure and function of reovirus has been

reviewed (Joklik, 1981). Reovirus is a double stranded, non-enveloped, RNA virus

surrounded by double concentric icosahedral capsid shell which contains transcriptase and

methyl transferase as an integral part of the viral core (Spandidos and Graham, 1976). The

migration pattern through polyacrylamide gel during electrophoresis revealed that reovirus is

made up of 10 genomic segments, namely large (L), medium (M) and small (S) numbered 2,

3, and 4, respectively (Gouvea and Schnitzer, 1982; Hrdy et al., 1979; Spandidos and

Graham, 1976; Shatkin et al., 1968) and 4 non-structural proteins (microNS, sigma NS, p17,

and p10) (Bodellon et al., 2001). Additionally, there are at least 10 viral proteins (Ni and

Kemp, 1995; Varela, 1994) and each of which shows different genomic segments (Varela,

1994). Similarly, the protein encoded by avian reovirus has three classes namely; λ (large), µ

(medium) and δ (small). The S1 segment contributes to the infective nature of reovirus (Ni

and Kemp, 1995; Weiner et al., 1977). Subsequently, a polymorphism was observed among

12

reovirus isolates within the same serotype (Wu, et al., 1994; Rekik 1990; Gouvea and

Schnitzer, 1982b; Hrdy et al., 1979).

Fig. 1.1. Avian Reovirus particle (from Connolly, J. L. and T. S. Dermody. 2002).

Mode of infection

Horizontal and vertical transmissions are two possible routes of infection for avian

reovirus. It has been well established that the horizontal mode of reovirus transmission

occurs through direct exposure to feces (Kerr and Olson, 1964; Jones, 1972 and 1978;

Robertson, et al., 1984). Another mechanism is through vertical transmission, whereby, the

progeny acquire virus from the dam while the ova are in situ (Hussain et al., 1981; Menendez

et al., 1975; Van der Heide and Kalbac, 1975; Desmukh and Pomeroy, 1969b). Glass (1973)

showed the possibility of egg transmission. This form of disease transmission occurred at a

level much lower than that horizontal. Menendez and co-workers (1975) were able to show

egg transmission of reovirus by inoculating 20 breeders with FDO-1 isolates through nasal,

oral, and esophageal routes, and subsequently, three birds tested positive for reovirus

(Menendez et al., 1975). Van der Heide (1975) challenged a low dose of reovirus

13

subcutaneously into breeders and later detected infection in eggs laid between 8 and 12 days

post inoculation. In a related study (Al-Muffarej, et al., 1996) showed that both the trypsin

resistant and non-resistant strain of reovirus was transmitted to eggs although at a different

rate, the former having the higher (19.2%) rate.

Pathogenesis

It has been reported that in reovirus pathogenesis, both the intestine and bursa (Jones,

1989; Kibenge, et al., 1985) serve as the portal of entry and site of initial virus replication. At

12-24 hours after oral inoculation, virus particles can be detected in these organs.

Afterwards, it spreads to other tissues/organs through the circulatory system. Two days after

infection regardless of site of experimental inoculation (Ni, et al., 1995), virus can be

detected in the liver and spleen, and the number of virus particles peaks at 4-6 days after

challenge. Ni, et al. (1995) noted that reovirus replicates primarily in the intestines and can

spread to other organs. Avian reovirus causes enteritis or malabsorption, but when it spreads

to other organs, it induces viral arthritis. Both malabsorption and viral arthritis can be found

in the same bird. According to several studies, the liver appears to be the primary target

organ for reovirus replication (Jones, 1989; Kibenge, et al., 1985; Mandelli, 1978). However,

there appears to be greater virus expression in the duodenum than in the liver, and it is likely

that much of the virus load is shed through the feces (Kibenge et al., 1985).

It has been implied that mobile cells, i.e., macrophages facilitate the spread of

reovirus (Kibenge et al., 1985; Tang et al., 987). Additionally, macrophages from reovirus-

infected chickens are primed to produce nitric oxide in response to T cell cytokines and

bacterial lipopolysaccharides (Pertile et al., 1996; Neelima et al., 2003).

14

It has been reported that reovirus infection is deemed to be age-linked and dose-

dependent (Jones, 1985; Roessler and Rosenberger, 1989; Meanger, et al., 1997). It was

pointed out that age-associated infection (Roessler and Rosenberger, 1989) might be due to

improved ability of the bird’s immune system to prevent virus dissemination and in lower

cellular virus content in all of the affected tissues. Clinical disease tends to be more

pronounced in birds challenged at 1 day of age compared to challenge at two weeks of age

(Rosenberger et al., 1989). Repeated oral exposures to the virus can lead to virus persistence

in the intestines compared to a transitory influence associated with a single oral inoculation

(Jones, 1994). Highly pathogenic isolates (2408, S1733) cause extensive cellular damage

compared to a low pathogenic isolate (2177) (Rosenberger, et al., 1989). Meanger et al

(1997) confirmed that high challenge doses of the three reovirus isolates resulted in more

severe lesions. Olson (1959) suggested that the inflammatory process during reovirus

infection persists even when no virus can be detected.

Virus replication

For reovirus replication to occur, cellular apoptosis is important, and in order for this

to happen, virus attachment of the protein sigma1 to a cell surface receptor is necessary

(Tyler et al., 1995). Apoptosis is the cellular host response to infection (O’Brien, 1998), and

it is directly induced by reovirus infection both in vivo and in vitro (Labrada et al., 2002;

Connolly et al. 2002). In vitro, the cytopathic effect (CPE) induced by reovirus is manifested

by cell shrinkage, rounding, detachment from plate, nuclear damage, chromatin condensation

(Fig. 2), which usually occurs 8 hours post infection and plateaus around 16 hours (Labrada

et al. 2002). Thus, the reovirus CPE involves both apoptosis and syncitium formation

(Bodelon et al., 2002). The S1 genome segment of reovirus is responsible for the syncytium-

15

inducing property of the virus (O’Hara et al., 2001; Duncan et al., 1988) through its encoded

10kDa (p10) fusion protein (Shmulevitz et al., 2000; Bodelon et al., 2000).

Fig. 1.2 Electron microscopic appearance of uninfected cell (A) Infected cells undergo various stages of chromatin condensation (B), margination of chromatin at the nuclear membrane (C) and complete condensation of the nucleus (D). Courtesy of Tyler et al., 1995; Journal of Virology.

Following infection, reovirus particles specifically protein δ1 ( Fig. 1.2) (Labrada et

al., 2002; Frazier et al., 1990, Matinez-Costas et al., 1997), attaches to the cell surface sialic

acid (Barton, et al., 2001) and JAM (Connolly et al., 2002). Protein δ1 is a filamentous

lollipop-shaped molecule about 48 nm in length, with a flexible "tail," approximately 40 nm

long by 4 to 6 nm wide, and terminates at its distal end in a globular "head", which consists

of the carboxy-terminal domains containing the receptor-binding sites folded into compact

globular conformations (Frazier et al., 1990). Apoptosis is characterized by virus-receptor

engagement, fusion with or penetration of cellular membranes, and disruption of host cell

transcriptional and translational machinery (Everett, et al., 1999). Reovirus receptors do not

initiate the signaling events that elicit apoptosis from the cell surface, but rather from

endocytic vesicles (Connolly et al., 2002). Viral uncoating is a major requirement for

16

programmed cell death induction (Labrada et al., 2002). This takes place at the endolysomes

in the cytoplasm. For example, when researchers treated the cultured cell with ammonium

chloride at the earlier onset of infection, viral protein replication was inhibited (Connolly et

al., 2002). During viral uncoating the outer capsid protein σ3 is removed, followed by

proteolytic cleavage of the inner capsid proteins µ1 and µ1C to form δ and φ, and

conformational changes in σ1. Consequently, this leads to the formation of infectious

subvirion particles (Connolly et al., 2002). Early transcription of the double stranded RNA is

encoded by viral polymerase. Transcription and translation of genome segments takes place,

and RNAs are then conservatively transcribed leading to synthesis of (+) sense mRNAs.

Fig. 1.3 Reovirus replication. From http://www-micro.msb.le.ac.uk/335/Diarrhoea.html

These transcripts leave the core particle and are translated in the cytoplasm. The capsid is

assembled and the particles are released (Fig.1.3) (Connolly et al., 2002).

17

Virus Detection Several diagnostic tests have been developed to detect the presence of reovirus in

infected birds. In cultured cells, the CPE is evident at 72 hours post-challenge. For the

standard serological testing employed in the detection of reovirus, serum neutralization (SN)

is a commonly used technique (Giambrone, 1988; Olson, 1975). SN can detect specific long

lasting antibodies, but the method requires the use of live virus and cell culture. For large

sample sizes, the ELISA assay for detection of reovirus can be used. The ELISA results

closely correlate with the serum neutralization results (Liu et al., 2002). A variation of the

ELISA assay is the monoclonal capture ELISA test (Pai, 2003; Chen et al., 2004; Liu, 2000).

This technique is more sensitive than conventional ELISA since it detects the target viral

RNA. For example, the ELISA results obtained when using the expressed sigmaC and

sigmaB proteins were found to be 100% correlated with serum neutralization and that

between serum neutralization and conventional ELISA, it showed about 89% correlation

(Shien et al., 2000; Liu et al 2002). In this thesis, the expressed sigma proteins were used as

coating antigens in inducing the neutralization of antibodies against reovirus. Sigma proteins

are the targets for type specific neutralizing antibodies (Wickramasinghe et al., 1993). The

sigmaB protein is the major component of the outer capsid while the sigmaC is the part that

attaches to the cell (Schnitzer, 1982). Therefore, specificity is apparent. Additionally,

molecular techniques were developed (Liu et al., 1999) such as in situ hybridization (ISH)

and reverse transcriptase in situ polymerase chain reaction (RT-in situ-PCR), although the

RT in situ PCR test was more sensitive and provided the rapid, sensitive, and specific

detection of avian reovirus infections compared with the ISH. Earlier, indirect fluorescent

18

antibody testing (IFA) was applied to tissue sections or impression smears to determine the

presence of viral antigens (Adair, et al., 1987).

Prevention

Avian reovirus is naturally infective in domestic fowls, but the mere presence of the

virus is not indicative of an infectious condition. Induction of an infectious condition is

dependent upon numerous events in the bird responding to the alteration of the host

environment. To assure prevention of infectious bouts of reovirus infection, poultry

producers must adhere to strict biosecurity practices such as thorough cleaning and

disinfection of poultry houses after each grow out cycle. Since reovirus can survive for at

least 10 days on feathers, wood shavings, chicken feed and eggshells (Savage et al., 2003), it

is important that these materials be removed from the poultry houses when clean-up and

disinfection is done.

Vaccination is one of the most important tools for prevention. This has led to

development and usage of vaccines, live and inactivated (Van der Heide, 1983). It is aimed

at providing direct immunity to chickens through active immunization either by

administering the vaccine at a young age or to breeders, who pass antibodies to their chicks

via the egg. Passive immunity facilitated by maternal vaccination allows for the transfer of

mainly immunoglobulin G (IgG) antibody via the eggs. These IgGs are sequestered from the

maternal blood and transported into the yolk mass and can cross the embryonic yolk sac

membrane into the embryonic circulation (Dohms et al., 1978; Roth et al., 1976; Kramer et

al., 1970). Therefore, by vaccinating the breeders, egg transmitted diseases such as those

caused by reovirus may be blocked (Van Loon et al., 2001). Breeder vaccination against

19

tenosynovitis resulted in immunity of progeny against experimental reovirus challenge

compared to unvaccinated control (Van der Heide et al., 1976). Recently, in ovo

administration of an antibody complex vaccine was studied (Guo et al., 2002). Given at day

18 of incubation, the antibody-complex vaccine provided at least 70% protection and

apparently did not affect hatchability. With regard to available breeder pullet vaccination

programs, it was reported that, day old progeny from hyperimmunized pullets receiving one

dose of live and 2 doses of inactivated reovirus vaccine provided the highest numerical

antibody titer and best resistance to clinical infection following experimental challenge

(Giambrone, 1986). However, in one study, multiple vaccinations of chickens with reovirus

strain RAM-1 produced a broadly specific neutralizing antibody response, but the protective

immunity against challenge seemed to be type-specific (Meanger et al., 1997)

Organs Associated with the Digestive Tract

Liver

Avian liver is subdivided into a left and a right lobe that is connected cranially by a

bridge dorsal to the heart (Dyce et al., 1996). The right lobe is larger than the left lobe. The

chicken liver weighs about 35-51g representing 1.7-2.3 % (relative weight) of the body

weight (Nickel et al., 1977). The largest part of the liver is located in the part of the body

enclosed by ribs while the remainder lies in the sternum. It lies against the heart,

proventriculus, gizzard and cranial end of the duodenal loop, the spleen and the gall bladder.

Hepatocytes and mesenchymal cells comprise the largest cellular mass in the liver.

The importance of the liver to the metabolic activity of the body can be seen in large

quantities of nutrients and other substances it receives via the portal vein from the gut

20

(Nickel et al., 1977). The body cannot directly utilize many of the substances absorbed from

the intestinal tract, and therefore, those substances must be converted or otherwise stored in

the liver before being released into the circulation. The liver is involved in numerous

important functions in the metabolism of protein and lipid (Geraert et al., 1996; Belloir et al.,

1997; Griffin et al., 1992; Leveille et al., 1975), and carbohydrates and vitamins in addition

to its vital role in the detoxification of body wastes and other toxins (Nickel et al., 1977).

The endocrine function of the liver is to synthesize proteins for gradual release into the

bloodstream instead of storing them in the cytoplasm as secretory granules. These protein

granules are produced by the hepatocytes.

Another type of cell present in the liver consists of the phagocytic cells of the

reticuloendothelial system and is known as Kupffer cells, which are liver resident

macrophage cells (Chang et al., 1996). These cells secrete cytokines such as interleukin-1

(IL-1), IL-6, and tumor necrosis factor α (TNF) (Kaiser, 1996), which are involved in

synthesis of acute phase reactants. In poults exhibiting PEMS, IL-6 and IL-1 production were

increased as a result of the ability of macrophages to produce these cytokines despite the

impaired ability of the cells to phagocytized foreign antigens (Heggen-Peay, 2002). In

addition, during hepatic injury the extent of the cellular damage can be determined by the

blood alanine aminotransferase (ALT) and blood aspartate aminotransferase (AST) activities

(Chang et al., 1996).

While the liver is the principal organ where insulin-like growth factor I (IGF-I) and

IGF-II are derived (McNabb, 2000), their production by a variety of extrahepatic tissues

suggests both autocrine and paracrine modes of action in addition to typical endocrine

mechanisms (Le Roith et al., 1992). In agreement with this concept, IGF-I gene expression is

21

not detected in the liver until hatch (Kikuchi et al. 1991), which may mean the circulating

IGF-I is extrahepatic in origin in the developing avian embryo (McMurtry, 1998). Avian

IGF-I has been purified and sequenced, and it was found to contain 8 amino acid

substitutions in comparison with human IGF-I (Ballard et al., 1990). On the other hand IGF-

II was found to contain 13 amino acid residues that differed from human IGF-II, and six of

these 13 differences occurred in the C-domain sequence (Kallincos et al. 1990). Insulin

regulates the transcription of these related growth factors (Houston and O’Neil, 1991) that

are structurally and functionally similar with that of the IGFs (Lewitt, 1994). In addition,

IGFs actions are mediated by surface receptors which in turn are mediated by the IGF

binding proteins (IGFBP). These IGFs were found to be important in growth, development

and metabolism in birds (McMurtry et al., 1997). In both chickens and turkeys, IGF-II is

relatively higher than IGF-I during embryo development and declines with time (McMurtry

et al., 1998). IGF-I is important during posthatch skeletal muscle development (Conlon et al.,

2002). In PEMS infected poults, depression of both of these factors was reported (Doerfler et

al., 2000). Similarly, in a study by Leili et al. (1997), prolonged feed restriction led to a

progressive growth retardation, wherein body-weight gain and bone growth were reduced.

The plasma concentrations IGF-I and IGF-II were decreased, and the degree of reduction in

the plasma concentrations of these growth factors seemed to be directly proportional to the

magnitude of feed restriction.

Likewise, fasting, protein deprivation, and insulin deficiency depressed the levels of

both IGF-I and IGF-II (McMurtry, 1998; McMurtry, et al 1997; 1996). According to Kita

(1996), IGF-I and not IGF-II is affected by plane of nutrition in chickens. But then, it was

later suggested that plasma IGF-I dramatically increases after withdrawal of feed and return

22

to normal level after refeeding (Mc Murtry, et al., 1998). And that IGF-II responds

differently to nutritional state in chicken either it is feed withdrawal against feed restriction.

The action of IGFs at cellular levels depended on their concentration in the circulation.

Endocrine Glands

Pituitary Gland

The pituitary glands or hypophysis lies at the base of the brain below the

hypothalamus, and is subdivided into two parts, the adenohypophysis (glandular part) and

neurohypophysis (neural part). Unlike mammals there is no pars intermedia in birds (Scanes,

1986). In chickens, differentiation of pituitary glands from Ratkhe’s pouch occurs at day 10

of embryonation (Sturkie, 2001). This reddish brown organ weighs about 10-22mg in hens

and 11-25mg in cockerels and about 13-36mg in capons (Nickel et al., 1977). It is the major

endocrine gland as it influences the secretion of hormones from several other glands.

Typically, cells in the pituitary gland are classified according to the type of hormones they

secrete, e.g., a) somatotrophic b) mammotrophic/lactotrophic c) gonadotrophic d)

thyrotrophic and e) corticotrophic cells (Bossis et al., 2004; Mikami et al., 1984). The

secreted hormones act on target organs, tissues, or cells by interacting with specific receptors

found cell surfaces, within the cell’s cytoplasm, or nucleus (Sturkie, 2001).

The posterior pituitary gland on the other hand, is made up of the neurosecretory

terminals for the secretion of mesotocin (MT) or arginine vasotocin (AVT) (Scanes, 2000).

AVT is an antidiuretic hormone and is the counter part of arginine vasopressin in mammals

while MT is the oxytocin principle. AVT modulates different aspects of reproductive

23

behavior including courtship vocalization and copulation (Jurkevich et al., 2003) in addition

to its role in osmoregulation and adaptation processes during the perihatch period (Takahashi

et al., 2003; Grossman et al., 1995). MT is decreased during acute heat stress (Wang et al.,

1989). Infusion of MT has no effect on cardiovascular functions, plasma ions and osmolality

(Robinzon et al., 1988), but it does participate in some regulatory effects on the blood flow to

the kidneys as renal perfusion during hemorrhage is positively correlated with plasma MT

levels (Bottje et al., 1989).

Pancreas

The pancreas is a long, narrow, grey-white gland that lies in the mesentery connecting

the duodenal loop (Hodges, 1974), it consists of dorsal and ventral lobes which are connected

distally (Dyce et al., 1999). In chickens, the notochord is the source of chemical signals

required for pancreatic development (Seung et al., 1997). The ablation of the notochord at

stage 11 of embryonation leads to the absence of gene expression needed for pancreatic

differentiation (Seung et. al., 1997). The avian pancreas is divided into two major and

functionally distinct areas; the endocrine pancreas, which consists of hormone producing

cells and exocrine cells which secrete enzyme involved in digestion (Chang et al., 1996;

Hodges, 1974). The exocrine pancreas, which is the larger part of the pancreatic tissue,

secretes the pancreatic juice through the zymogen granules in acinar cells. The enzymes are

carried into the duodenum via the ducts (Nickel et al., 1977; Hodges, 1974). On the other

hand, the pancreatic islets or the islets of Langerhans perform the endocrine role through the

alpha, beta, delta and pancreatic polypeptide producing cells (PP cells) (McNabb, 2000;

Sitbo et al., 1980).

24

Alpha cells secrete the hormone glucagon whereas the beta cells are involved in the

production of insulin; these two hormones are antagonistic to each other (Figs. 1.4-1.5).

Insulin is a protein, which is made up of two chains (alpha & beta) of amino acids connected

by a disulfide bond (Hazelwood, 1986). It mainly increases transfer of plasma glucose and

other monosaccharides across the cell membrane. Additionally, insulin plays a role in

glucose oxidation, glycogenesis, proteogenesis (Teruel et al., 1998), lipogenesis (Dupont et

al., 1999), and ketogenesis. Insulin is formed first as production proinsulin. Insulin has the

ability to reduce glycogenolysis with accompanying incorporation of glucose molecules into

de novo production of glycogen in the liver (Hazelwood, 1986).

The primary activity of the pancreatic alpha cells, which produce glucagon, a 29-

amino acid peptide, is to counter the effects of insulin and promote glycogenolysis and

gluconeogenesis (Hazelwood, 2000; Sitbon et al., 1980). Pancreatic glucagon circulates in

the plasma at about 2-4ng/ml plasma normally and increases in the presence of fasting

(Hazelwood, 2000). In broilers suffering from spiking mortality syndrome and exhibiting

hypoglycemia, it was found that the mean pancreatic glucagon content was depressed

98.75% when compared to normoglycemic control birds (Davis, 1995). Both insulin and

glucagon are secreted mainly from the pancreas, but with a 99% surgical removal of the

pancreas both hormones could be produced from extrapancreatic sources (Hazelwood, et al.,

1989), i.e., the splenic remnant tissue. Additionally, only the PPA was eliminated in the

circulation when the pancreas was removed (Cocla et al., 1982). Glucagon is reported to be a

stress hormone (Freeman, 1980), and consequently, it is responsible for activating the

hypothalamo-hypophyseal-adrenocortical axis (Freeman, 1980). This was concluded after

glucagon was injected intra-abdominally into day old chicks, which responded after 7 days

25

with increased adrenal weights and adrenal cholesterol stores. The chicks then became

hypoglycemic and hypercholesteraemic (Freeman, 1980). Nonetheless, the glucagon/insulin

ratio is essential to the regulation of plasma glucose level in birds since increases in the ratio

leads to diabetes (Sitbon et al., 1980)

In birds suffering from malabsorption of nutrients, depressed glycogen storage is

evident (Davis, 1995). During stress responses glycogen is converted to glucose by glucagon

(glycogenolysis) to augment plasma glucose level. Therefore, chicks with S1133 infection

are likely to become hypoglycemic, especially during the time when they suffer from the

extreme effects of reovirus infection.

Fig.1.4. Metabolic effects of glucagon

26

Fig. 1.5 Metabolism of insulin

Thyroid

In developing embryos, the thyroid gland arises as a single midventral evagination of

the floor of the pharynx before dividing into two separate lobes by day 5 of incubation

(Astier, 1980). Then between 10.5 and 12.5 day of embryonation, functionality of the thyroid

axis occurs (Lui et al., 2003). In the adult, it is situated on either side of the trachea, very

close to the carotid artery and the jugular vein. These two lobes appear as round to oval in

shape and are enclosed in a thin connective tissue capsule and are dark red in color

(Wentworth et al., 1986). This red color appearance is attributed to the dense vascular

network of the parenchyma (Astier, 1980) that distinguishes thyroid from its neighboring but

27

similarly shaped organ, the last thymic lobe (Dyce et al., 1996). Furthermore thyroid gland

comprises about 0.2% of the total body weight of chickens (Duke, 1997).

Cells within the thyroid glands are arranged in chord like rows (McNabb, 2000),

whose height depends on the thyroid activity (Khan et al., 1999). Actively secreting cells are

tall columnar cells with vacuolated colloid, but it can be reduced in height, i.e., cuboidal or

low columnar and colloids can accumulate in quiescent thyroids (Astier, 1980). The colloid

in follicles that are surrounded by the secretory cells of the thyroid is a gelatinous substance

composed of an iodinated protein, the thyroglobulin (TG). Anatomically, avian thyroid does

not have calcitonin cells or parafollicular cells in the interstitial cells, and this is due to the

fact that the ultimobrachial gland which is the origin of calcitonin secreting cells is not

connected with the thyroid gland throughout the life span of birds (Astier, 1980; Wentworth

et al., 1986)

Dietary iodine is converted into iodide (I-) before its absorption from the small

intestines before its release into the circulation (Engelking, 2000). Iodine concentration in

thyroid is proportionate to the amount of dietary iodine intake (Newcomer, 1978). In general,

iodine metabolism involves 3 major steps leading to secretion of hormones, i.e., 1.) iodide is

actively transported into the thyroid by the follicular cells, also known as iodide trap, 2) a

peroxidase system within the thyroid converts iodide to I2 and then to I+, and 3) a second

enzyme system is responsible for combining the iodinated tyrosines with in the polypeptide

chain of TG to eventually form triiodothyronine (T3) and thyroxine (T4) (Wentworth et al.,

1986). The T4 and T3 are distributed throughout the body via the circulation and under the

control of thyroid-stimulating hormone (TSH) and are maintained across a large range of

28

physiological concentrations (Reyns et al., 2001). Thyroid hormone secreted from the thyroid

is mostly T4 (McNabb, 2000; Astier, 1980; Lam et al. 1986)).

Upon hatching the level of T3 in chickens increases until 2 weeks of age and

gradually decreases (Kuhn et al., 1993), but plasma T4 levels start rising at around day 15

(Thommes et al., 1977). Kuhn et al. (1993) noted that a positive correlation exists between T3

levels and growth. In young birds, this hormone is essential for yolk sac resorbtion,

functional maturation of the lungs, pipping, and hatching (Van der Geyten, 1997). T3 has

been reported to directly stimulate growth and maturation of embryonic chick cartilage and

enhance the in vitro action of somatomedins on cartilage growth (King, 1984). At the time

when the chick embryo switches from chorioallantoic to lung respiration, as it penetrates the

air sac, both plasma T3 and T4 reach their highest values (Lui et al., 2003).

Thereafter, during the growing period, thyroid hormones stimulate growth and

maturation (Liu, et al., 2004; Kahn et al., 1998). In relation to this, as chickens grows older,

the secretion of thyroid hormone is governed by the negative feed back mechanism involving

the hypothalamic-pituitary-thyroid axis (Engelkin, 2000). This mechanism is also seen when

exogenous T3 is supplemented in the diet and hyperthyroidism develops as indicated by

dramatic increases in plasma T3 level and liver T4 conversion declines (Collin et al., 2003). In

addition, the plasma T3 concentration in adult birds is lower whereas the growth hormone

and T4 are high depending on the strain of birds (Kuhn et al., 1993). In certain conditions

such as the malabsorption syndrome, the thyroid is the earliest target organ (Rudas et al.,

1986), wherein the T3 and T4 levels were decreased as a result of oral inoculation of infected

intestinal homogenates. In the same manner, hypothyroidism greatly affects growth during

the post hatch period whereby body weight gain is affected more than bone growth (King et

29

al., 1984). Meanwhile, it has been suggested that feed restriction in chickens resulted in high

levels of circulating T4 and decreased T3 levels, which means that the availability of T4 from

the liver and thyroid is not affected (Van der Geyten, 1999; Reyn et al., 2002; Geris et al.,

1999; Rosebrough et al., 1989; Kuhn et al., 1987; Klandorf and Harvey 1985). As a

consequence, since liver is the main contributor of T3, decreased hepatic iodothyronine

deiodinase type II (ID2), which converts T4 into T3, and increased hepatic iodothyronine

deiodinase type III (ID3), which converts T3 into reverse T3 (rT3) and T2 will reduce the T3

supply to the whole body and decrease energy metabolism. It has long been known that

thyroid hormones exert a profound effect on development, growth, and metabolism of

skeletal tissues (Klaushofer et al., 1995). This is due to the interactions between T3, the most

active form, and nuclear receptors that bind to thyroid hormone response elements, causing

modification of the expression of specific genes associated with growth and development.

Apart from the effects of thyroid hormones in growth and maturation (Darras et al.,

2000), it aids in thermogenesis specifically a T3-driven thermogenic effect (Collins et al.,

2003; Silva, 1985). In fact, T3 plays a major role in metabolic heat production that is

necessary for maintenance of high and constant body temperature (McNabb, 2000; Darras et

al., 2000). Thus, defeathering results in increase thyroxine level in plasma and experimental

chickens that are defeathered have higher metabolic rates as well as increased thyroid mass

(Pietras, 1981; Tulett et al., 1980).

30

Adrenals

Adrenal glands are paired yellowish-brown, oval to triangular shaped structures

situated at the cranial pole of the corresponding kidney. They are related ventrally to the

ovary or epididymis (Dyce et al., 1996). Similar to mammals, the adrenal gland cells arise

from the neural crest and mesoderm (King et al., 1984). In chickens, there is no clear

distinction between outer cortex or inner medulla unlike the arrangement in mammalian

adrenals, which is distinctly separated. In birds, the chromaffin cells are intermingled with

cortical cells (Harvey et al., 1986). In the adrenals, there are two types of chromaffin cells,

which secrete epinephrine and norepinephrine (Astier, 1986). Functional activation of the

adrenal gland in chick embryo is regulated at three different levels- 1) the adrenal gland

itself, 2) the anterior pituitary, and 3) the hypothalamus or what is commonly termed as

hypothalamo-pituitary-adrenocortical (HPA) axis. From day 8 until shortly after day 14 of

embryonic development, adrenal gland appears capable of secreting glucocorticoids

independently, at which point the pituitary influences adrenocortical activity and at the same

age, the hypothalamic level of control also starts (Jenkins et al., 2004; Bossis et al., 2004).

Avian adrenals account for two regions of steroidogenic (cortical) tissue; the

subcapsular zone (SCZ), which secretes aldosterone and the inner zone (IZ) producing the

corticosterone (Holmes et al., 1984; Klingbeil et al., 1979). These cells from the SCZ

consisted of irregular nuclei, mitochondria with shelf-like cristae and a moderate abundance

of smooth endoplasmic reticulum. The IZ cells contain more vascular space and contain large

round nuclei surrounded by an abundance of smooth endoplasmic reticulum and the

mitochondria have tubular rather than shelf-like cristae (Klingbeil et al., 1979; Pearce et al.,

31

1978 and 1979). Corticosterone is the primary secreted hormone of the adrenals (Carsia et

al., 1987; Harvey, et al., 1986).

Adrenal size is influenced by factors such as response to seasonal and environmental

change, onset of reproduction, population densities and social interaction and in some species

habitat (Holmes et al., 1980). Adrenals are essential for the increase in free fatty acids

induced by insulin (Veiga et al., 1983). The adrenal relative weights of feed restricted birds

tend to be increased but decrease with time (Carsia, 1998; Freeman, 1981, Nir, 1975). Water

deprivation can cause adrenal corticosterone to increase (Tome, 1985). Stressors that induced

corticosterone secretion include hyperthermia (Edens and Siegel, 1975), anesthesia (Scanes

et al., 1980), infections (Edens et al., 1991; Curtis et al., 1980), exertion (Rees et al., 1984),

immobilization (Beuving and Vonder 1979) and frustration (Harvey et al., 1985). Davis et al.

(1989) observed that feed deprivation for 3 days on newly hatched poults resulted in a low

corticosterone level, and eventually, mortality was observed. This was in contrast to findings

in chickens (Edens et al., 1991c) that infection coupled with 24 hour fasting lead to increase

plasma CS. Adrenocortical hormone secretions are stimulated by adrenocorticotrophic

hormone (ACTH) (Koscis et al., 1989; Carsia et al., 1987), angiotensins and other putative

regulators (McNabb, 2000).

Current Study

Avian reovirus infection causes significant physiological and pathological problems

in chickens and turkeys. Understanding the mode of disease transmission can help in the

design of managerial programs that may alleviate or even prevent the disease process. It is

important to evaluate the physiological impact or changes that underlie the disease. Thus, this

32

investigation dealt with the pathogenicity of PEMS associated agents, passive antibody

transfer from hyperimmunized breeders, and experimental in ovo disease transmission. In

addition, for the very first time, this study dealt with the potential vertical transmission of

PEMS-associated turkey reovirus ARCU98 using an in ovo approach in broiler chickens.

33

CHAPTER 2

Comparative Pathogenicity Study of PEMS Associated Astrovirus and Reovirus

in the Presence or Absence of E. coli in Turkey Poults1

ABSTRACT. Poult enteritis and mortality syndrome (PEMS) is an acute, contagious enteric

disease characterized by high morbidity and mortality of poults between 2-4 weeks of age.

The current study was conducted to compare the relative pathogenicity of various organisms

(viruses and bacteria) associated with PEMS, given alone or in combination through oral

gavage to turkey poults under a controlled environment. Determinants for clinical pathologic

changes were body weight, liver, bursa, spleen and thymus relative weights. In one

experiment, poults were challenged with the PEMS astrovirus (Tast-OSU, 0.2mL, 103

EID50), Tast-OSU + E. coli (0.2mL, 1 x 108cfu), and E. coli alone. The results of this

experiment showed that the 7 day post-challenge body weight gain was significantly less in

all treatment groups with the Tast-OSU group exhibiting the poorest weight gain. Thymic

(P<0.05) and bursal atrophy were observed in Tast-OSU group. In another experiment, poults

were challenged with the reovirus ARVCU98 (0.2mL, 103 EID50), Tast-OSU (0.2mL, 103

EID50), or ARVCU98 +Tast-OSU given at day one post hatch followed three days later with

the E. coli challenge (0.2mL, 1 x 108cfu) and a sham-exposed control group. The results of

this experiment showed a progressive and significant decrease in body weight at six days

after infection. The poults exhibited severe watery, foul smelling, foamy diarrhea with thin-

walled, gaseous distended intestines. Bursal and thymic atrophy were observed at nine days

after infection with no difference in the liver and spleen weight. Taken together, these

experiments showed that while no mortalities occurred, the astrovirus and reovirus reduced

34

body weight and induced bursal and thymic atrophy. The addition of E. coli with these

viruses increased the severity of the weight loss and associated signs of PEMS.

Keywords: Poult enteritis and mortality syndrome, Reovirus, Astrovirus, E. coli, Poults

INTRODUCTION

Poult enteritis and mortality syndrome (PEMS) emerged as a disease with an

unknown etiology in 1991 that caused a highly significant economic loss in the poultry

industry in the US, particularly the southeastern region. The disease affected poults between

two and four weeks of age, but some resistance developed as maturity approached. PEMS

primarily affected the digestive tract as poults exhibited severe diarrhea with associated

immune dysfunction as evidenced by its effect on the bursa of Fabricius and thymus. The

fecal-oral route appeared to be the main mode of transmission of the disease.

Several organisms have been linked to PEMS, but to date it is classified as a disease

with multifactorial etiology. Certain viruses, bacteria, insects, protozoans as well as some

environmental factors contributed to the ontogeny of the disease. One of the viruses

implicated in PEMS was the reovirus ARVCU98. ARVCU98, which was isolated from fecal

filtrates, caused increased incidence of thymic hemorrhaging, gaseous intestines, decreased

liver size, and altered liver AST and ALT activities (Heggen-Peay et al., 2002a). Earlier

reports also implicated astrovirus in enteric diseases in turkey poults (Koci et al., 2000;

Reynolds, 1986). Turkey astrovirus can induce body weight loss, thymic atrophy, and

diarrhea (Koci et al., 2003; Qureshi et al., 2001; Yu et al., 2000). Additionally, it has been

established that a small round virus (SRV) isolated from the same fecal pool as the reovirus

35

ARVCU98 was actually a turkey astrovirus (Tast-OSU) and was the same as the SRV that

had been described earlier (Qureshi et al., 1999, 2001; Yu et al 2000).

In this investigation, the turkey astrovirus- Tast-OSU (Qureshi et al., 1999, 2000,

2001) and turkey reovirus- ARVCU98 (Heggen-Peay et al., 2001), which were isolated from

a fecal pool from PEMS-positive, turkey coronavirus-negative poults, were examined to

compare their pathogenicity in the development of PEMS. The viruses were given by oral

gavage either alone or followed in combination with E. coli (Edens et al., 1997a, b) three

days post infection (DPI). The pathogenicity of the viruses either alone or in combination

with E. coli was evaluated by monitoring their effects on body weight, lymphoid organ and

liver weights, and over all performance of poults at different time points after infection.

MATERIALS AND METHODS

Animal Welfare. This study was conducted following the guidelines established by the

North Carolina State University (NCSU) Institutional Animal Care and Use Committee.

Poults. Specific pathogen free poult embryos1 were used in these experiments to provide

intestinal homogenates for control poults. Commercial poults were the source of

experimental animals. Hybrid strain poults were used in trial 1 of experiment 1, and Nicholas

poults were used in trial 2 of experiment 1. Within six hours after the poults hatched, they

were transported to North Carolina State University, chosen randomly for wing-banding,

weighed and placed into treatment groups for each experiment. In each of the two

experiments, initial ambient temperature was started at 35C and was decreased by 4C at

intervals of 7 days. Continuous incandescent lighting was provided in each experiment. Feed

and water were provided ad libitum. In experiment 1, the standard North Carolina

36

Agriculture Research Service (NCARS) turkey starter diet fed. In experiment two, a special

irradiated feed2, equivalent to the standard NCARS turkey starter diet, was used to prevent

bacterium or viral contamination. Feed and water for the poults in experiment 2 were placed

in bulk into the isolators and weighing scales were also placed in the isolators until the end of

the investigation. Poults removed from the isolators were first caught using the glove ports in

the isolators, placed into an antechamber that was opened and resealed before a second seal

was removed from the isolator to allow collection of the sample poults. After the sample

poults had been removed from the antechamber, it was resealed and fumigated with a

chlorine compound.

Experiment 1. For experiment 1, there were two trials consisting of four treatment groups

that are listed as follows: 1) control poults received an intestinal homogenate from normal

specific pathogen free (SPF) poult embryos, 2) turkey astrovirus isolated at Ohio State

University (Tast-OSU; 103 EID50), 3) E. coli (1x108cfu/poult), 4) Tast-OSU + E. coli. Forty-

one poults per group were raised in Petersime3 brooder batteries. At seven days post hatch,

the poults were challenged orally with 0.2mL of the virus challenge material. Three days

post virus challenge when the poults were 10 days of age; group 3 and 4 poults were

challenged orally with E. coli.

Experiment 2. For experiment 2, three treatment groups were established as follows: 1)

Cornell University avian reovirus isolate (ARVCU98, 103 EID50) + Tast-OSU (103 EID50), 2)

ARVCU98 + Tast-OSU + E. coli, 3) control challenged with phosphate buffered saline, pH

1 SPF poults were obtained from Ohio Agriculture Research and Development Center, Wooster, OH. 2 Feed irradiated

37

7.0 (PBS). There were 25 poults per group, and the poults were maintained in bubble

isolators4 with HEPA-filtered, heated air. On the day of hatching, each poult was gavaged

orally with virus challenge inoculum and treatment 2 was given E. coli at 3 days post virus

challenge.

Body weights. All poults were weighed before placement into their treatment groups. In

experiment 1, body weights were determined 7, 10, 13, 15 and 17 days after virus challenge.

In experiment 2, body weights were determined at 4, 6, 9 and 11 days after virus challenge.

Lymphoid organs. The following organs, bursa of Fabricius, spleen and thymus (all lobes

from the left side of the neck) were removed and weighed at each time point listed above. A

total of three to ten poults per treatment were sampled each time body weights were

determined as shown above. The organ weights were converted to percent of body weight

and reported as lymphoid organ relative weights. For study 2, the same protocol was

followed with the addition of the determination of the liver relative weight.

Statistical Analysis. A completely randomized experimental design was utilized for both

experiments. The data gathered were analyzed using the General Linear Models procedures

of SAS (SAS Institute, 1999). There were treatment and time main effects, and the treatment

X time interaction was included in the statistical model. If the interactions were not

significant, their degrees of freedom were incorporated in the residual error term. If there

were significant main effects, treatment means were separated using the least significant

difference procedure of SAS (1999). Significance was set at P≤ 0.05.

3 Petersime Equipment Co., Gettysburg, OH 45328 4 Standard Safety, Chicago, IL.

38

RESULTS

Experiment 1

Trial 1. Tables 2.1 and 2.2 depict the BW of conventional poults at 7, 10, 13, 15 and 17DPI

that were challenged either with Tast-OSU, Tast-OSU + E. coli, E. coli alone, or SPF turkey

embryo intestinal homogenate (control). In trial 1, at 17DPI a significant BW depression

(P<0.056) was observed in Tast-OSU challenged poults. E. coli poults were comparable to

the control. The BW of the virus challenged poults was consistently lower in comparison

with the control. When the 7 day weight gain was calculated, Tast-OSU challenged birds had

lower weight gain than the control, but the Tast-OSU + E. coli group was not significantly

different from the control.

Trial 2. In trial 2 (Table 2.2), at 10 days after virus challenge, Tast-OSU + E. coli and E. coli

had significantly (P = 0.002) lower body weights than control and Tast-OSU groups. The

seven day body weight gain of Tast-OSU- and E. coli-challenged groups differed from

control and Tast-OSU + E. coli (P = 0.068).

The lymphoid organ integrity results for trial 1 are shown in Table 2.3. At 3 days post

E. coli challenge, the bursa of Fabricius relative weights of poults in the control and both

Tast-OSU-challenged groups were significantly lower than E. coli alone. At five days after E.

coli challenge, only the Tast-OSU challenged groups were showing depressed relative

weights of the bursa of Fabricius. At seven days after E. coli challenge, there were no

differences among treatment groups for bursa of Fabricius relative weights. The relative

weights of the thymus from Tast-OSU challenged groups approached significance (P = 0.07)

39

at five days after E. coli challenge but not at any other time. The relative weights of the

spleen were not affected by either virus or bacterial challenge.

In trial 2 of experiment 1, the relative weights of neither the bursa of Fabricius nor the

spleen were affected by either the virus or bacterial challenges (Table 2.4). The low thymus

relative weights of Tast-OSU-challenged poults approached significance at three days after

E. coli challenge, and at seven days after E. coli challenge, the thymus relative weights of

Tast-OSU virus challenged poults and E. coli challenged poults were significantly (P< 0.05)

lower than controls.

Experiment 2

Table 2.5 illustrates the mean body weights of poults oral challenges of combined

pathogens [ARVCU98 + Tast-OSU, ARVCU98 + Tast-OSU +E. coli and or PBS (control)]

associated with PEMS. At six days after challenge, poults receiving the combined challenges

had mean body weights that were significantly less (P <0.0001) than control and persisted

with significantly lower body weights than controls at nine (P = 0.08) and eleven (P =

0.0011) days after challenge.

In experiment 2, the relative weights of neither the spleen nor the liver were affected

significantly by the combined pathogen challenges (Tables 2.6A-2.6D). Inconsistency

marked the responses of the thymus and the bursa of Fabricius to the combined pathogen

challenges (Tables 2.6A-2.6D). The relative weights of the bursa of Fabricius of poults in the

ARVCU98 + Tast-OSU were reduced significantly at four days after the challenge. At eleven

days after ARVCU98 + Tast-OSU and ARVCU98 + Tast-OSU + E. coli, the relative weights

of the bursa of Fabricius were reduced significantly compared with control. The relative

40

weight of the thymus was reduced significantly (P = 0.0049, 0.001, and 0.0301) by

ARVCU98 + Tast-OSU and ARVCU98 + Tast-OSU + E. coli challenges at six, nine and

eleven days, respectively, after challenge compared with control.

DISCUSSION

In an earlier report, Tast-OSU (formerly called SRV) given either orally or through

contact exposure to seeder poults led to growth depression starting at 2-3 days post exposure

and caused a severe foamy diarrhea (Qureshi et al., 1999). Tast-OSU caused alteration in the

lymphocyte subpopulations and reduced lymphoblastogenic response to T cell mitogens.

Likewise inoculation to young poults resulted in 11% mortality. When turkey coronavirus

was given in combination with Tast-OSU, mortality rate was doubled. It was concluded that

Tast-OSU when given alone or in combination with other viral agents can be detrimental to

turkey poult under controlled conditions and possibly exacerbated in field exposures.

Therefore, because PEMS is considered to be a condition with a multifactorial

etiology, experiments were undertaken to investigate the effects of combinations of putative

etiologic agents implicated in the development of PEMS. In experiment 1 of this study, Tast-

OSU was examined in combination with E. coli isolates found associated frequently with

PEMS (Edens et al., 1997 abc). Results in Table 2.1 show that Tast-OSU and Tast-OSU + E.

coli were able to depress the weight gain of poults. E. coli by itself did not cause a depression

in weight gain, and this was in contrast with earlier findings (Edens, 1997 a). However, the

conditions of the current investigation were different from the original study by Edens et al.

(1997a) who used a more stressful protocol. All E. coli-challenged poults developed a severe

diarrhea similar to PEMS and mortality was increased by both atypical E. coli colony types

41

when the poults were made immunodeficient (Edens et al., 1997a). Heggen-Peay (2000)

reported that the atypical E. coli by themselves did not cause any detrimental effect on poult

performance. E. coli in combination with Tast-OSU caused 7 day body weights to be lower

than control (P=0.068) but not E. coli alone. These observations suggest that each component

etiology for PEMS must play a specific role and that independently, none are as potent as

combinations.

Similar trends were seen in trial 2 of experiment 1 (Table 2.2). Generally, the body

weights of the challenged poults were lower in comparison with the control. However, it is

important to note that body weights of the treatment groups at the time of E. coli challenge

were uneven, suggesting that some of the groups might have been compromised by other

unknown factors. Therefore, the 7 day weight gain differences of the challenged poults

compared with controls might be more important than periodic weight gain information. The

7 day weight gain data showed that Tast-OSU and E coli treatments alone were responsible

for a significantly decreased weight gain (P=0.045) at 7 days after E. coli challenge. Tast-

OSU + E. coli was not different from control but had a numerically lower weight gain.

Even though the poults given the E. coli challenge in both trials 1 and 2 of this

experiment did not show a dramatic depression in weight gain either alone or in combination

with Tast-OSU, it must be considered that the challenge was given at 10 days post hatch.

During the first 10 days of development, the enteric microbiota of the turkey poult is

maturing and conceivably may have developed climax populations of commensal bacteria

that have the potential to inhibit colonization of many different bacteria including the PEMS-

associated E. coli. The data presented here do not detract from the original PEMS-associated

bacteria reports (Edens et al., 1997abc). In this study there was a clear depression in body

42

weight gain at 3dpi when E. coli was given alone in trial 1, and in trial 2, there was an overall

depression in 7dpi weight gain that was as severe as the depression caused by Tast-OSU

challenge. Instead, these observations point to the potential that the PEMS-associated E. coli

might be able to exert if given during a time when the poult might be more susceptible, such

as day of hatch rather than after several days of development after hatching.

In experiment 1, the E. coli challenge did not result in depression of either bursa of

Fabricius or spleen relative weights. Tast-OSU challenge did cause a significant (P=0.02)

depression in relative weight of the bursa of Fabricius at 5 days after E. coli challenge. In

trial 1 at 5 days after E. coli challenge, thymus relative weights in the Tast-OSU and Tast-

OSU + E coli challenged poults were reduced (P=0.07) by 31.3% and 25%, respectively,

when compared to the control. In trial 2, thymic atrophy was observed at 3 (Tast-OSU only)

and 7 days (Tast-OSU, Tast-OSU + E. coli, and E. coli) (P <0.0001). Qureshi et al (1999)

reported thymic atrophy (30.8%) in poults challenged with Tast-OSU between 4 and 8 DPI.

The lymphoid organ relative weight data show that Tast-OSU either alone or in combination

with E. coli can cause bursal and thymic atrophy in young turkey poults.

Heggen-Peay et al. (2001) reported that PEMS-associated reovirus ARVCU98,

depressed the body weights of experimental poult between 22% and 28%. The lymphoid

organ (bursa of Fabricius and thymus) relative weights were decreased in comparison with

the control poults and that there was an increase in the incidence of thymic hemorrhaging. A

similar trend was observed in experiment 2 of this investigation.

In experiment 2, body weights were depressed in turkey poults given combinations of

viruses (ARVCU98 + Tast-OSU) and viruses plus E. coli (ARVCU98 + Tast-OSU + E. coli).

Body weight depression evident as soon as 6DPI and persisted through 11DPI. These

43

observations clearly demonstrated the effect of E. coli when given together with reovirus

because it exacerbated the body weight depression even in a controlled environment. Thus, it

is possible that when combinations of several etiologic agents associated with PEMS are

present, the possibility of development of fulminating PEMS is greatly increased.

In experiment 2, thymic atrophy in poults given both combinations of PEMS

pathogens was observed at 6 DPI and persisted through 11DPI (Table 2.6A-2.6D). Thymic

atrophy is a typical finding in PEMS (Qureshi et al., 1997). Schultz et al. (2000) reported that

when poults were exposed to thymic filtrate, PEMS-like symptoms were exhibited by those

poults. Therefore, it was concluded that filtrates of thymus tissues from PEMS-infected

poults appear to harbor etiologic agents that can cause development of PEMS. An astrovirus

was isolated from thymic filtrate (Schultz et al., 2000).

In this study, turkey astrovirus Tast-OSU was used in combination with the turkey

reovirus ARVCU98. Bursal atrophy was evident as early as 4DPI in ARVCU98 + Tast-OSU

poults and at and 11DPI in ARVCU98 + Tast-OSU and ARVCU98 + Tast-OSU +E. coli

groups. Relative weights of both bursa of Fabricius and thymus are depressed significantly

when poults are exposed to combinations of either reovirus, astrovirus or E. coli. The bursal

and thymus are the primary lymphoid tissues in birds, and when their integrity becomes

compromised, immunological functions likely suppressed in response to antigenic

challenges. Ultimately the loss of cellularity in the thymus and bursa causes alterations in

both T and B cell populations and in the mononuclear phagocytic system, which can result in

immunosuppression or immunodysfunction found in PEMS (Qureshi et al., 1997).

The relative weights of neither the spleen nor the liver were affected significantly by

the combinations of PEMS causing agents in experiment 2. When absolute liver weights of

44

the ARVCU98 + Tast-OSU +E. coli-challenged poults were measured, they were greatly

reduced in comparison with controls. The absolute weight effects were due to the smaller

body weights of the challenged poults.

In conclusion, it can be implied from the results of this investigation that exposure to

Tast-OSU and ARVCU98 can lead to body weight reduction and that the addition of E. coli

can further aggravate the situation. These etiologic agents, in combination appear to have the

potential to disrupt the immune system and cause the immunodysfunction that has been

reported for PEMS-infected poults (Qureshi et al., 1997). The results obtained in this

investigation were obtained in highly controlled environmental conditions. It is assumed that

under commercial conditions the responses to these etiologic agents would be exacerbated

and possibly results in fulminating PEMS cases.

45

45

Table 2.1. The effect of small round virus (Tast-OSU) and E. coli oral challenge on growth of conventional poults5 (Trial 1)

Challenge 1 76 10 13 15 17 7 day gain

g 3 Control2 114.3 139.50 208.3 248.0 303.5 164.8 +2.51a +4.02 b +9.38 a +15.6a +15.0a +11.1 a Tast-OSU 105 139.60 198.7 226.7 261.3 121.7

+2.65a +4.23 b +9.38 a +15.6a +17.3b +12.8b

Tast-OSU + E. coli 108.4 141.7 180.0 245.7 270.0 138.3 +2.65a +4.23 b +9.38 a +15.6a +17.3b +12.8 ab

E. coli 111.5 160.2 185.7 260.7 326.8 166.8

+2.51a +4.02 a +9.38 a +15.6a +15.0 a +11.1 a

Pvalue4 0.11 0.002 0.21 0.52 0.056 0.068

1Poults orally challenged with 103EID50 Tast-OSU at 7 days of age followed by 1X108E. coli (turkey isolate Type I and II at 10 days of age. 2Poults in control were challenged with 0.2ml of turkey embryo intestinal homogenate prepared from control (normal) SPF embryos. 3Represent mean body weight (g) 4Significance value as determined by General Linear Model in SAS 5Poults were Hybrid strain. 6Days in age

46

46

Table 2.2. The effect of small round virus (Tast-OSU) and E. coli oral challenge on growth of conventional poults5 (Trial 2)

Challenge 1 76 10 13 15 17 7 day gain

g 3 Control2 118.8 157.66 198.02 21.0 286.3 135.33a +3.92a +4.58 ab +10.74a +12.0a +15.0a +10.50 Tast-OSU 124.2 153.60 181.33 235.0 260.0 100.0b +3.72a +4.34 ab +10.74 a +12.0a +17.3a +9.09

Tast-OSU + E. coli 112.1 134.4 196.33 199.0 258.25 127.25a

+3.72a +4.34 c +10.74a +12.0a +17.3a +9.09 E. coli 116.0 146.9 191.3 229.3 240.0 99.50b

+3.72a +4.34 b +10.74a +12.0a +15.0a +9.09

Pvalue4 0.15 0.002 0.70 0.24 0.35 0.068

1Poults orally challenged with 103EID50 Tast-OSU at 7 days of age followed by 1X108E. coli (turkey isolate Type I and II at 10 days of age. 2Poults in control were challenged with 0.2ml of turkey embryo intestinal homogenate prepared from control (normal) SPF embryos. 3Represent mean body weight (g) 4Significance value as determined by General Linear Model in SAS 5Poults were Nicholas strain. 6Days in age

47

47

Table 2.3. The effect of small round virus (Tast-OSU) and E. coli oral challenge1 on relative organ weight of conventional poults 5 (Trial 1)

Age6 Organ Control2 SRV SRV+ E. coli E. coli Pvalue4

%3 13 Bursa 0.21 + 0.02 b 0.18 + 0.02b 0.19 + 0.02 b 0.31 + 0.02a 0.03

Thymus 0.17 + 0.02a 0.11 + 0.02a 0.15 + 0.02a 0.18 + 0.02a 0.16

Spleen 0.06 + 0.01a 0.07 + 0.01a 0.09 + 0.01a 0.08 + 0.01a 0.21

15 Bursa 0.24 + 0.01 a 0.17 + 0.01 b 0.20 + 0.01 ab 0.23 + 0.01 a 0.02

Thymus 0.16 + 0.01 a 0.11 + 0.01 b 0.12 + 0.01 ab 0.15 + 0.01 a 0.07

Spleen 0.09 + 0.01a 0.09 + 0.01a 0.09+ 0.01a 0.07 + 0.02a 0.33

17 Bursa 0.18 + 0.02a 0.12 + 0.02a 0.19 + 0.02a 0.18 + 0.02a 0.14

Thymus 0.13 + 0.02 a 0.11 + 0.02 a 0.12 + 0.02 a 0.18 + 0.02 a 0.10

Spleen 0.07 + 0.01 a 0.11 + 0.01 a 0.09 + 0.01 a 0.09 + 0.02 a 0.32

1Poults orally challenged with 103EID50 ARVCU98 at 7 days of age followed by 1X108E. coli (turkey isolate Type I and II at 10 days of age. 2Poults in control were challenged with 0.2ml of turkey embryo intestinal homogenate prepared from control (normal) SPF embryos. 3Represent mean body weight (g) 4Significance value as determined by General Linear Model in SAS 5Poults were Hybrid strain .6Age in days

48

48

Table 2.4. The effect of small round virus (SRV) and E. coli oral challenge on relative organ weights of conventional poults5 (Trial 2)

Age 6 Organ Control2 SRV SRV+ E. coli E. coli Pvalue4

%3 13 Bursa 0.20 + 0.02a 0.18 + 0.02a 0.20 + 0.02a 0.16 + 0.02a 0.55

Thymus 0.13 + 0.01 a 0.10 + 0.01 a 0.11 + 0.01 a 0.16 + 0.01 a 0.07

Spleen 0.08 + 0.01 a 0.07 + 0.01 a 0.06 + 0.01 a 0.09 + 0.01 a 0.30

15 Bursa 0.20 + 0.02a 0.21 + 0.02a 0.19 + 0.02 a 0.18 + 0.02 a 0.77

Thymus 0.08 + 0.01a 0.06 + 0.01 a 0.10 + 0.01 a 0.10 + 0.01a 0.35

Spleen 0.09 + 0.01a 0.09 + 0.01 a 0.09 + 0.01 a 0.07 + 0.01a 0.76

17 Bursa 0.22 + 0.02 a 0.20 + 0.01 a 0.22 + 0.02 a 0.19 + 0.01 a 0.40

Thymus 0.16 + 0.01 a 0.11 + 0.01b 0.10 + 0.01 b 0.10 + 0.01 b 0.00

Spleen 0.08 + 0.01 a 0.10 + 0.01 a 0.10 + 0.01 a 0.09 + 0.01 a 0.52

1Poults orally inoculated with 103EID50 SRV at 7 days of age followed by 1X108E. coli (turkey isolate Type I and II, (Edens et al., 1997) at 10 days of age. 2Poults in control were inoculated with 0.2ml of turkey embryo intestinal homogenate prepared from control (normal) SPF embryos. 3Represent mean body weight (g) 4Significance value as determined by General Linear Model in SAS 5Poults were Nicholas strain. 6 Age in days

49

Table2.5. The effect of ARVCU98, Astrovirus and E. coli oral challenge on body weight (g) of conventional poults

Days after challenge Treatment 0 4 6A 9 11 ( n=25) (n=25) (n=6) (n=6) (n=6)

Control 59.76 140 192+6.31a 266+11.26c 338+8.77s

ARV+Astrovirus 60.72 126 159+6.31b 227+11.26b 300+8.77b

ARV+Astrovirus 60.20 120 137+6.31c 246+11.26ab 280+8.77b

+E. coli Pvalue 0.0001 0.0805 0.0011

AMeans +SEM ab, In a column unlike letters differs significantly P<0.05

50

Table 2.6A. Relative organ weight (%) of challenged poults at 4 days post

inoculation Treatment ThymusA Bursa Spleen Liver

Control 0.12+0.01a 0.16+0.009a 0.07+0.01a 3.38+0.13a

ARV+Astrovirus 0.14+0.01a 0.08+0.009b 0.08+0.01a 3.15+0.13a

ARV+Astrovirus 0.09+0.01a 0.13+0.009a 0.07+0.01a 3.01+0.13a

+ E. coli Pvalue 0.1282 0.0002 0.7196 0.2042

AMeans +SEM ab, In a column unlike letters differs significantly P<0.05

51

Table 2.6B. Relative organ weight (%) of challenged poults at 6 days post challenge. Treatment ThymusA Bursa Spleen Liver

Control 0.13+0.01a 0.13+0.01a 0.06+0.008a 3.29+0.14a

ARV+Astro 0.09+0.01b 0.14+0.01a 0.07+0.008ab 3.02+0.14a

ARV+Astro 0.10 +0.01a 0.11+0.01a 0.09+0.008a 3.20+0.14a +E. coli Pvalue 0.0049 0.0979 0.0714 0.4403 AMeans +SEM ab, In a column unlike letters differs significantly P<0.05

52

Table 2.6C. Relative organ weight (%) of challenged poults at nine days post inoculation. Treatment ThymusA Bursa Spleen Liver

Control 0.15+0.008a 0.16+0.01a 0.07+0.007a 2.95+0.14a

ARV+Astrovirus 0.12+0.008b 0.14+0.01a 0.07+0.007a 3.10+0.14a

ARV+Astrovirus 0.09+0.008c 0.14+0.01a 0.08+0.007a 2.86+0.14a

+ E. coli

Pvalue 0.0010 0.4092 0.4693 0.5164 AMeans +SEM ab, In a column, means with unlike superscripts differ significantly P<0.05.

53

Table 2.6D. Relative organ weight (%) of challenged poults at 11 days post challenge. Treatment Thymus Bursa Spleen Liver

Control 0.15+0.01a 0.31+0.01a 0.10+0.01a 3.01+0.14a

ARV+Astro 0.11+0.01b 0.13+0.01b 0.10+0.01a 3.21+0.14a

ARV+Astro 0.11 +0.01b 0.14+0.01b 0.14+0.01a 3.07+0.15a

+ E. coli Pvalue 0.0301 0.0001 0.2455 0.5909 AMeans +SEM ab, In a column unlike letters differs significantly P<0.05

54

CHAPTER 3

Comparative Pathogenicity of PEMS-Associated ARVCU98 and the Field-Associated S1733 Reoviruses on Broilers Inoculated in ovo.

ABSTRACT. The effects of in ovo inoculation of two reoviruses- ARVCU98 (turkey) and

S1733 (chicken)- in broiler chickens as shown by changes in body weights, relative weights

of liver and lymphoid organs, and blood plasma chemistry were examined to characterize

the pathogenicity of these isolates. Two independent trials were conducted using two

separate groups of embryonated eggs. Eggs at day 9 of embryonation were challenged via the

yolk sac with 100µL/egg (ARVCU98 at 103 TCID50; S1733 at 108 EID50). Percent

hatchability for the control group was 70%, but hatchability for ARVCU98 was 24% for

S1733 (1:100 and 1:500) treatment groups hatchability was 59% and 67%, respectively.

Body weights of virus-challenged chickens were significantly less (P≤0.05) than controls at 7

and 14 days of age. The bursa of Fabricius, thymus, and liver relative weights were

decreased. Blood plasma glucagon and insulin decreased significantly (P≤0.05) in the virus-

challenged groups. The T3/T4 ratio of virus-challenged chickens was slightly decreased

compared with the control but were not significant. The lower levels of plasma insulin and

glucagon coupled with a slight depression in the T3/T4 ratio suggested a metabolic

dysfunction in virus-challenged chickens. The IGF-I and IGF-II levels in virus-challenged

chickens were slightly but not significantly higher than the control. Cellular integrity tests, as

indicated by aspartate amino transferase (AST) and amino alanine transferase (ALT)

activities suggested that S1733 had an effect from 1 to 14 days of age but in the ARVCU98

challenged group, there was only a one day change in the activities of these enzymes.

Immunohistochemical examination did not reveal viral antigen on either bursa or thymus at

55

14 and 28 days of age. Electron microscopic examination of the lymphoid organs and several

endocrine glands revealed virus-related changes in cellular ultrastructure. Using electron

microscopy, the reoviruses were found in the liver and pancreas. These results demonstrate

that both the ARVCU98 and S1733 can be transmitted in ovo from day 9 of incubation. The

PEMS-associated ARVCU98 can inhibit growth and development in broiler chicken body

weight as seen in earlier studies with turkey poults and might be pathogenic in chickens

under field conditions

Keywords: Poult enteritis and mortality syndrome, reovirus, broiler, endocrine responses

56

INTRODUCTION

Avian reovirus infection is classified as an important economic disease of poultry

worldwide. One of the agents implicated in poult enteritis and mortality syndrome (PEMS)

that affects young turkey poults is an avian reovirus, ARVCU98. PEMS was first known as a

disease with unknown etiology in the early 1990’s in North Carolina. It was widely

characterized as an acute, highly transmissible, infectious enteric disease with high morbidity

and mortality rate and affected poults failed to reach target market weight. Other signs of

PEMS are diarrhea, dehydration, lethargy, and lymphoid organ and liver pathology. There is

an increase in the pro-inflammatory cytokines by activated macrophages in poults infected

with PEMS, which has been thought in part to be responsible for the intestinal inflammation,

gut motility and anorexia (Qureshi et al., 1997; Heggen, et al., 2000). Therefore, is generally

immunosuppressive in nature.

To date, no single organism has been found to be the causative agent for PEMS, and

PEMS which is regarded as a disease with a multifactorial etiology. Reovirus, ARVCU98,

was found to cause significant physiological and pathological changes turkey poults (Chapter

2), and when it was given in combination with a turkey astrovirus (Tast-OSU) and atypical E.

coli (Chapter 2), significant physiological changes similar to PEMS were observed. In the

area of North Carolina where PEMS developed, there is also a very high concentration of

broiler and broiler breeder farms. Because reovirus can be introduced into a facility by

mechanical means, one must be concerned that there might be some dissemination of virus

from chickens to turkeys or from turkeys to chickens. Therefore, in this investigation, the

potential for the turkey reovirus, ARVCU98, to be transmitted vertically to chickens was

57

examined. The objective of this investigation was to determine if in ovo administered

reoviruses, ARVCU98 and S1733, had effects post hatch in broiler chickens.

MATERIALS AND METHODS

Animal Care. This project was approved and conducted under the supervision of the North

Carolina State University Animal Care and Use Committee which has adopted Animal Care

and Use Guidelines governing all animal use in experimental procedures.

Virus. The frozen (-20°C) stock S1733 reovirus isolate (supplied by Intervet USA, Inc.,

Millsboro, Delaware) had a 105.8, 50% embryo infective dose (EID50)/mL in a 1:100 dilution.

The PEMS-associated ARVCU98 reovirus had a 103 TCID50 titer at 72 hr post inoculation at

day 9 of incubation (Heggen-Peay, 2001).

Embryo Inoculations and Incubation. Two separate trials were conducted using virus-

challenged embryonated eggs at day 9 of incubation. Embryonated broiler breeder eggs were

obtained from the North Carolina Agricultural Research Service flock (Ross X Arbor Acres),

which had not been vaccination against reovirus. Eggs were maintained under standard

incubation and hatching conditions until they hatched. Eggs were candled to assure viability.

Eggshells were thoroughly disinfected with a 70% ethyl alcohol wash followed by an

antibiotic (Pen/Strep) wash. A small hole was then drilled on the small end of the egg and

100µL of virus inoculum/egg was injected into the yolk sac. The inoculation site was sealed

with paraffin wax. Inoculated eggs were returned to the incubator until day 18 of incubation

when they were segregated into their various treatment groups and transferred to pedigree

hatching baskets. When the treatment groups hatched, they were wing banded and weighed.

58

On the hatching date, blood was collected from 5 birds/treatment group from which plasma

was collected and stored at -80°C until used for plasma chemistry analysis. Day old chickens

were placed into electrically-heated Petersime (Petersime Equipment Co., Gettysburg, OH

45328) brooder batteries. The control and virus-challenged chickens were maintained in

different but identically-controlled isolation rooms. Body weights were recorded at days 1, 7,

9, 14 and 28, and randomly caught chickens were killed by carbon dioxide asphyxiation on

days 14 and 28 for determination of the lymphoid organ (bursa, thymus and spleen) and liver

relative weights. Tissue samples from the anterior pituitary, pancreas, thyroid and liver were

collected for transmission electron microscopy. Tissue samples from the thymus and bursa

were gathered for viral antigen detection and for histopathology (hematoxylin and eosin B).

Animal Husbandry. Hatched chickens were placed into heated brooder battery pens (15

chickens/pen) by treatment group. Pen temperature was maintained at 32°C for one week,

reduced to 27°C at week two and then to room temperature (24°C) at week three through

four weeks of age. Feed and water were provided for free consumption.

Body and organ weights. At 14 and 28 days of age, live body weights were determined and

the chickens were killed by carbon dioxide asphyxiation. The chickens were then necropsied,

and liver and lymphoid organs weights were recorded for calculation of relative weights

{(organ wt [g])/(BW [g]) X 100}.

Feather scoring. Feather tracks on the back, neck, wing, thigh, breast area and abdomen

were graded subjectively and composite whole body feathering scores were calculated

(Edens, 1998). Whole body feather scores ranged from 1 (poorest) to 5 (best).

Serum AST and ALT. Blood was collected in sterile serum separation tubes from five

chickens per treatment group at 1, 14 and 28 days of age. Tubes were refrigerated overnight

59

for expression of serum. Quantitative determination for serum amino alanine transferase

(ALT) and aspartate amino transferase (AST) activities were conducted following the

manufacturer’s protocol (Stanbio Laboratories, Boerne, Texas)

Blood Plasma Chemistry. At 21 and 28 days of age, blood for plasma was collected via the

jugular vein into sodium EDTA from 5 chickens per treatment group. Blood samples were

centrifuged, and plasma was collected and stored at -20°C until assayed. Plasma for

glucagon analysis was stored in vials containing aprotinin (Sigma-Aldrich, St. Louis , MO).

Plasma radioimmunoassay for glucagon (Linco, Inc., St. Charles, MO), insulin (McMurtry,

et. al., 1983), thyroxine (T4) and triiodothyronine (T3) (McMurtry, et. al. 1988), insulin-like

growth factor (IGF-I) (McMurtry et al., 1994), IGF-II (McMurtry, et al., 1998) were sent to

USDA-ARS, Beltsville, MD and assayed in the laboratory of Dr. J. P. McMurtry.

Reovirus ELISA. Detection of reovirus antibodies from the maternal serum was assayed

using the FlockChek (IDEXX) reovirus ELISA test kit.

Immunohistochemistry. Bursa of Fabricius and thymus were collected from the three

treatment groups at 14 and 28 days of age. Organs were embedded in optical cutting

temperature compound (OCT) (Tissue Tek; Miles., Elkhart, IN), frozen and cut into 5µ

sections. Tissue sections were deparaffinized and incubated for 30 minutes with convalescent

serum made in PBS and heat inactivated at 56ºC and was used as the primary antibody.

Convalescent sera were obtained from chickens from each treatment group at 14 days of age.

After a series of washes in phosphate buffered saline (PBS), the slides were incubated with

1:15 dilution of FITC–conjugated goat anti-chicken immunoglobulinG (IgG) heavy and light

(H&L) chains (Southern Biotechnology Associates Inc., Birmingham, AL) for 30 minutes

60

and later washed with PBS. Slides were examined under fluorescent microscopy. Slides

were scored as positive or negative for the presence of reovirus antigen in a given tissue.

Transmission Electron Microscopy (TEM). Adrenals, liver, anterior pituitary, pancreas

and thyroid were cut into 1mm3 block and fixed in 3% glutaraldehyde and 0.1 M Na

cocadylate buffer, pH 7.4 at 4ºC. Samples were rinsed in 3 cold 30 minutes changes of 0.1M

Na cocadylate buffer, pH 7.4. After which samples were post-fixed for 2 hours at 4ºC in 1%

osmium tetroxide in 0.1 M Na cocadylate buffer, pH 7.4.After samples were washed as

described above, they were rehydrated with a graded ethanol series from 30-100% ethanol

alcohol (EtOH). Tissues samples were embedded in BEEM capsules with fresh 100% Spurr’s

solution overnight at 70ºC after they were washed 3X with 100% Spurr’s before infiltrating

with 1:3, 1:1, 3:1 Spurr’s:EtOH. Tissues were then thick-sectioned at 1µ and stained with

Toluidine blue for preliminary examination under light microscopy before thin-sectioning at

75-100nm and affixed to 200-mesh uncoated grids. Thin sections of tissues were stained for

1 hour with 4% uranyl acetate and 4 minutes with Reynold’s lead citrate at room temperature

prior to examination under TEM at 80kV (JEOL JEM-100S). The TEMs were digitized and

processed using Adobe Photoshop 7.0 (Adobe Photo Systems, Inc. San Jose, CA 95110-

2704).

Histopathology. Bursa of Fabricius and thymus were fixed in 10% buffered formalin.

Tissues were dehydrated, embedded in paraffin, sectioned at 5µm and stained with

hematoxylin and eosin B. Tissues sections were examined under light microscope, and

photomicrographs were made with a Leitz photomicroscopy system using Kodak T160 slide

film.

61

Statistical Analysis. A completely randomized experimental design was used. The data were

analyzed using the General Linear Models procedures of SAS (SAS Institute, 1999). There

were treatment and time main effects and treatment X time interaction in the statistical

model. If the interactions were not significant, their degrees of freedom were incorporated in

the residual error term. If there were significant main effects, treatment means were separated

using the least significant difference procedure of SAS (1999). Significance was set at P≤

0.05.

RESULTS

Hatchability and post-inoculation mortality. The overall hatchability of control eggs was

70%. Eggs challenged with ARVCU98 had 24% hatchability compared to the 59% and 67%,

respectively, for the 1:100 and 1:500 dilutions for the S1733 treatment groups (Table 3.1).

Embryo viability at 18d of incubation was 53% for the control, 37% for the ARVCU98, and

42% and 52%, respectively, for the two S1733 groups. Before 7d of age, only one chicken in

the ARVCU98 group died, but there were no deaths in the other groups.

Body weight and growth. Body weights of the broiler chickens in Trials 1 and 2 (1, 7, 9, 14

and 28 d of age) are given in Tables 3.2 and 3.3, respectively. Even though there was a

significant trial difference for body weights, trends within the two trials were similar. In trial

1, the hatching body weights were less than the hatching body weights in trial 2. In trial 1,

there were no significant differences among treatment groups for hatching body weights

(Table 3.2), but in trial 2 day of hatch body weights of S1733-challenged (1:500) chickens

were heavier than all other groups (Table 3.3). At 7 d of age, the S1733-challenged chickens

had significantly lower body weights than the controls in both Trials 1 and 2, and the

62

ARVCU98 chickens had significantly lower body weights in trial 2. At 14 d of age, the body

weights of all virus-challenged groups were significantly less (P<0.05) than controls, and

ARVCU98 chickens had the lowest body weights among all the groups (Tables 3.2 and 3.3).

At 28 d of age, there were no significant differences among the treatment groups even though

the virus-challenged groups had lower body weights (Table 3.2 and 3.3). Using average daily

gain (Table 3.4) as an indicator of virus influence on growth, it was found that in ovo virus

challenge caused a significant decrease in weight gain (P <0.05).

Feed conversion. Among treatment groups, feed conversion ratios (FCR) were not

significantly different (Table 3.5).

Lymphoid organ and liver weights. Tables 3.5A-3.5B and 3.6A-3.6B show the 14 and 28d

of age relative weights for liver, bursa of Fabricius, thymus, spleen and liver in trials 1 and 2,

respectively. The relative weights of the liver from virus-challenged chickens were

decreased significantly in trial 1 only (Table 3.5A). At 28d of age, liver relative weight from

S1733 (1:100) challenged chickens was less than the liver relative weights of chickens given

either ARVCU98 or S1733 (1:500), but all of the relative liver weights of the birds given

virus challenges were greater that the relative weight of the controls (Table 3.5B). Among the

relative weights for the bursa of Fabricius, thymus and spleen, there were no significant

differences due to virus challenge (Tables 3.5AB and 3.6AB). In trial 1, the relative weight

of the bursa of Fabricius was less than virus-challenged groups at 14d of age, but at 28d of

age, the relative weight of the bursa of Fabricius of the S1733 (1:100) group was less than the

relatives weights of the bursae from all other groups. In trial 2, relative weights of the bursa

of Fabricius were not affected by any of the virus challenges (Tables 3.6AB). In trial 1, the

thymus relative weights of the ARVCU98-challenged birds at 14d and 28 d of age were less

63

(P=0.067 and P=0.016, respectively) than relative weights from other challenged groups and

control. In trial 2, virus challenge did not affect relative weight of thymus as compared with

controls.

Feather Scoring. Subjective scoring of feathering was performed (Table 3.7). Control

chickens were scored as 5 while virus challenged chickens, both ARVCU98 and S1733, were

assigned a feather score of 3.

Gross lesions. Upon necropsy, the virus-challenged groups exhibited thin-walled, gas-filled,

frothy intestines (Table 3.8). Noticeably, the S1733-challenged chickens had swollen,

hyperemic kidneys and hearts were enlarged in association with pericarditis in comparison

with the broiler chickens from ARVCU98 and control treatment groups.

Histopathology. Slides of bursa of Fabricius and thymus were prepared and examined under

light microscopy to determine the histopathological changes attributed to in ovo infection. A

summary of the histopathological signs in the bursa of Fabricius and thymus from reovirus-

infected chickens at 14d and 28d of age are presented in Tables 3.9A and 3.9B for trials 1

and 2, respectively. Eosinophilia, based on the presence of eosinophil-like cells, containing

spherical eosinophilic granules and bilobed nucleus, was a characteristic of virus infection in

both the bursa of Fabricius and the thymus and was evident with ARVCU98 and S1733

infection at 14d and 28d of age (Tables 3.9 AB; Fig. 3.4 to 3.15). The presence of

eosinophilia in the bursa and the thymus was also associated with loss of cellularity at both

14d and 28d of age (Tables 3.9 AB; Fig. 3.4 to 3.15). Macrophage infiltration into the

thymus and bursa of Fabricius was obviously increased when the chickens were challenged

with the reoviruses from both chicken (S1733) and turkey (ARVCU98) origin (Tables 3.9

AB; Fig. 3.4 to 3.15).

64

Blood Plasma Chemistry

Plasma Glucagon, Insulin, and Glucose. The blood plasma chemistry results are shown in

Table 3.10. There were significant differences among treatment groups for the plasma

glucagon (P ≤ 0.05) and plasma insulin (P = 0.0077) concentrations, but plasma glucose

concentrations were not affected by infection (P = 0.1079). Virus-challenged chickens had

significantly lower plasma glucagon than did control chickens. Plasma glucagon

concentrations in ARVCU98-, S1733 (1:100)-, and S1733 (1:500)- challenged chickens were

45%, 41, and 32% lower, respectively, than control concentrations. Plasma insulin

concentrations were not elevated as expected based upon the decreased plasma glucagon

concentrations but were instead decreased in virus challenged chickens (Table 3.10). The

plasma insulin concentrations in the ARVCU98- and S1733 (1:100)-challenged groups were

significantly lower (P≤ 0.0077) than control and S1733 (1:500)-challenged groups.

Insulin-like Growth Factors and Thyroid Hormones. The plasma insulin-like growth

factors (IGF-I and IGF-II) were not affected by virus challenge in these trials (Table 3.10).

Likewise, the plasma T4 and T3 concentrations were also not affected significantly by virus

challenge (Table 3.10). The T3/T4 ratio was also not affected significantly by virus infection

(Table 3.10). However, the plasma T3 and T4 concentrations were very low in comparison

with published values for these hormones.

Cellular Integrity. Cellular integrity of liver and muscle was also compromised by virus

infection as indicated by activity of two enzymes, amino alanine transferase (ALT) and

aspartate amino transferase (AST) that leak into plasma when there is cellular trauma due to

such cellular events as oxidative stress. Results shown in Table 3.11 reflect the fact that both

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ARVCU98 and S1733 in ovo challenge increased activities of these enzymes in the plasma.

S1733 caused an increase in ALT at hatching and all virus treatments caused an increase in

ALT at 14 d of age. There were time-dependent changes in ALT and AST activities (Table

3.11). The increased enzyme activities at 14 d of age corresponded to the time when the

virus-challenged chickens showed a spike in post-hatch mortality. From 14 to 28 d of age,

there is an apparent recovery as suggested by the decrease in the plasma activities of AST

and ALT in the virus-challenged chickens.

Immunohistochemistry. Tissue samples of the bursa and thymus (14 and 28 days of age),

treated with primary and secondary antibodies and examined microscopically did not reveal

evidence for viral antigens in the virus-challenged groups. However, examination of the

slides prepared for histopathology revealed numerous changes in the organization of the two

primary lymphoid tissues (Fig. 3.4 to 3.15). Summaries of these pathological changes are

presented in Tables 3.9A and 3.9B.

Transmission Electron Microscopy. Transmission electromicrographs (TEM) of pancreas,

anterior pituitary, and thyroid of control and reovirus-challenged broiler chickens at 14d of

age were examined (Fig. 3.16-3.27). In the TEMs representing thin sections through the

pancreas (Fig. 3.16-3.18), significant ultrastructural changes in acini cells in associated with

virus infection were observed. In control pancreas (Fig. 3.16) the cisternae of the cellular

reticulum was well developed and appeared to be actively involved in production of enzymes

for secretion as zymogen granules into the digestive tract. The zymogen granules were not

bounded by membranes. Mitochondria were well-formed and abundant. The endoplasmic

reticulum was widely dispersed within the cytoplasm. In pancreas from broilers challenged

with ARVCU98 reovirus, the cellular ultrastructure was radically different from the control

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(Fig. 3.17). The mitochondria were enlarged and appeared to be in many instances in a

degenerated condition. The endoplasmic reticulum in the acini cells was condensed with very

small cisternae indicative of low activity, which was supported by the observation of reduced

numbers of zymogen granules in the cytoplasm as compared with control chicken pancreas

acini cells. The nuclei of the acini cells also appeared to be very irregular and possibly in a

degenerative state. The pancreatic acini cells from S1733 reovirus-challenged presented

another very different ultrastructural appearance (Fig. 3.18). Under light microscopic

examination it was apparent that the cytoplasm of acini cells was literally packed with

zymogen granules. In the TEMs, it was apparent that the zymogen granules were

significantly smaller than the granules found in control and in ARVCU98-challenged acini

cells. The mitochondria in these acini cells were also condensed and widely dispersed. The

nuclei of the cells were generally large and diffuse as compared with those found in control

and ARVCU98-challenged pancreatic acini cells. The representative TEMs for the anterior

pituitary are presented in Figures 3.19 to 3.21. In thick sections and in the TEMs, it was

possible to distinguish two cell types based on staining properties, but there was no method

for distinguishing their true identities. One cell type was light staining and the other was dark

staining, and based upon these observations, the designation of chromophobe-like (light

staining) and chromophil-like (dark staining) was ascribed to these two cell types. In the

representative TEMS (Fig. 3.19 to 3.21), chromophobe-like and chromophil-like cells are

identified. In the control TEM (3.19) the cytoplasm of the chromophil-like cells had more

secretory granules than the chromophobes. Mitochondria were numerous and well defined in

both cell types, but occasionally in the chromophil-like cells mitochondria appeared to be

swollen and disrupted in control. In the representative TEM from the ARVCU98-challenged

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chicken, the density of the secretory granules in the chromophil-like cells (Fig. 3.20) was

apparently less than in control (Fig. 3.19).Less secretory granule density was suggested in

chromophobe-like cells also. Mitochondrial morphology was affected apparently by the

ARVCU98-reovirus challenge. They were enlarged in all cell types and appeared to be in a

degenerative state. The nuclei of the cells in the anterior pituitary of the ARVCU98

challenged chickens also were more irregular in shape than those found in control. The

morphological appearance of the chromophobe-like and chromophil-like cells in the anterior

pituitary of the S1733 reovirus-challenged chickens (Fig. 3.21)was different from both the

control and ARVCU98 reovirus-challenged chickens (Fig. 3.19 and 3.20). The

chromophobe-like cells and chromophil-like cells appeared to have very few secretory

granules in comparison whit similar cell types in control and ARVCU98 reovirus challenged

birds. The secretory granules in the chromophobe-like cells appeared to be larger and with

less dense staining properties. The mitochondria in the cell types in the S1733 reovirus-

challenged pituitary cells were greatly enlarged and appeared to be quite distorted in

comparison with control. Representative TEMs of the thyroid glands from control,

ARVCU98 reovirus-challenged, and S1733 reovirus-challenged chickens are presented in

Figures 3.22 to 3.24. Two features common to the reovirus challenged tissues were that the

mitochondria in the thyroid follicle epithelial cells were enlarged and distorted (Fig. 3.23 and

3.24), and the nuclei of the follicular epithelial cells were more irregular (Fig. 3.23 and 3.24)

than control (Fig. 3.22).

It was also possible to identify both the S1733 and ARVCU98 reoviruses in several

tissues of the challenged chickens. Shown in Figure 3.25 are numerous virus S1733 particles

in the liver of a challenged chicken. The virus appeared to be about 100nm in diameter. A

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unique TEM depicting ARVCU98 shedding from centroacinar cells into the lumen of the

acinus (Fig. 3.26). These virus particles appear to be around 80 nm in diameter. A

comparison of ARVCU98 reovirus and S1733 reovirus (in cross section) is presented in

Figure 3.27. Mitochondrial damage in these tissues was evident in the virus-challenged

chickens at 14 days of age. Also there was a dense population of zymogen granules in the

pancreas of S1733 challenged birds. In addition, in comparison to the control the follicular

cells of both the virus-challenged groups were taller in height or thicker. ARVCU98 was

demonstrated on the pancreas at 14 days of age, whereas S1733 was detected in the liver

(Figs. 3.35 and 3.26).

DISCUSSION

Earlier studies (Qureshi et al., 1997, Heggen-Peay et al., 2000) demonstrated that oral

inoculations of turkey poults with PEMS-associated reovirus (ARVCU98) caused lymphoid

organ and liver atrophy and decreased growth rate in turkey poults. In this report

pathogenicity of PEMS-associated ARVCU98 and a chicken reovirus isolate (S1733) were

investigated after virus-challenge inocula were given in ovo into the yolk sac of broiler

breeder eggs at 9 d of incubation. At 18 days of incubation (Table 3.1) percentage viability of

the virus-challenged eggs was lower in comparison with the control, and those injected with

ARVCU98 had the least viable eggs, which resulted in the lowest percent hatchability (24%)

at day of hatch. The low level of hatch-rate for the virus-challenged chickens might be due

to high virus replication in tissues that likely killed the embryos. These observations support

the contention of Menendez et al. (1975) and Van der Heide et al. (1983) that vertical

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transmission of reovirus was possible but at a low rate. The embryo lethality associated with

reovirus in eggs helps to explain the low rate of vertical transmission.

In ovo inoculation of ARVCU98 and S1733 viruses resulted in growth depression

from 7 d of age through 14 d of age with residual effects even through 28 d of age (Tables

3.2 and 3.3). In Trial 2, S1733 virus (1:500 dilution) at 1 d post hatch reduced body weight

(Table 3.3). The fact that a large percentage of the S1733 virus (1:500 dilution) reovirus-

challenged embryos developed and had hatch weights comparable with unchallenged

controls is somewhat surprising. The data suggest that there is, perhaps, a threshold for

lethality that was either inhibited by the embryo or that virus replication was more difficult

than expected when low numbers of the virus were injected. Motha (1987) challenged six-

day old embryonated eggs with a reticuloendotheliosis virus, and the hatchlings showed no

weight difference at day of hatch when compared to the unchallenged chickens.

Nevertheless, the results of this investigation show that reovirus given in ovo or by oral

gavage at hatch, based on previous PEMS investigations from this laboratory, can induce a

significant depression in post hatch body weight gain. Additionally, the average daily weight

gain (Table 3.4) was significantly less in the virus-challenged chickens. Although there were

no significant differences among treatment groups for feed conversion ratios (Table 3.5),

virus-challenged chickens appeared to be less efficient in converting feed to body mass.

Thus, impaired digestion, decreased uptake, and poor absorptive capacity in the intestinal

tract of the reovirus-infected chickens and turkeys will contribute to decreased weight gain

(Odetallah, 2001).

The relative weights of the bursa of Fabricius, thymus, and spleen were not affected

consistently by in ovo virus challenge or by the virus type (Tables 3.6A-3.7B). Sporadic

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effects were noted with both ARVCU98 and S1733 as they caused decreased relative weights

of the thymus but not the bursa of Fabricius. The reason for these inconsistent results could

be construed as an argument that other etiologic agents are required to allow for the

fulminating enteritis that is associated with PEMS in turkeys and of other enteritis problems

found in chickens. Liver relative weight of control chickens, as expected, was greater than

liver relative weights of virus challenged chickens (Tables 3.6A-3.7B). The results for

ARVCU98 reovirus challenge in chickens were comparable to those reported earlier in

turkey poults by Qureshi et. al. (1997) and Heggen-Peay (2001) who extended the

hypothesis that a chicken reovirus virus might have been involved in the etiology of PEMS in

the mid-1990’s. Support for this hypothesis comes from a report by Van der Heide (1980),

who found that day old chickens experienced high rates of mortality and liver necrosis when

challenged with a liver suspension from infected poults and that the turkey reovirus isolate

was neutralized in vitro by antiserum from chickens with a reovirus infection. It is very

possible that ARVCU98 reovirus might be a virus capable of infecting both chickens and

turkeys. Thus, it is possible that a reovirus of chicken origin might have been the progenitor

of the ARVCU98 that was involved in the index case for PEMS in 1991. The PEMS index

case was in an area of North Carolina that had a very high concentration of both chicken and

turkey farms in close proximity.

A plasma chemistry profile was established for chickens given an in ovo virus

challenged from either ARVCU98 or S1733 reoviruses. Plasma glucose levels were not

different among the treatment groups and tended to be somewhat higher (337-382

mg/dL)than reported nonfasted (210-250 mg/dL) values for chickens (Hazelwood, 2000).

This could be due to the fact that all the groups were full fed. Under a full feeding regimen

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with elevated plasma glucose, plasma glucagon is down regulated (Hazelwood, 2000). In

fact, plasma glucagon was at a level that was roughly 5-10% of chickens in a fasted state.

Plasma glucagon and insulin concentrations in ARVCU98 and S1733 (1:100) were

significantly lower in reovirus-challenged groups than in the control. Hazelwood (2000)

contends that glucagon in the domestic fowl is the more potent of the two pancreatic

hormones, but if that were to be the situation, the data would suggest that glucose levels

should have been less in the reovirus-challenged chickens even with lower plasma insulin

concentration (Table 3.11). Glucagon targets liver and muscle glycogen and converts it into

glucose. Liver atrophy was exhibited in S1733 reovirus-challenged chickens but not in

ARVCU98 which showed an increase in liver relative weight at 14 and 28 d post hatch.

Whether the virus challenge had affected uptake of plasma amino acids and glucose, used as

hepatic and muscle gluconeogenic substrates, was not determined in this investigation.

However, the decrease in insulin levels in reovirus-challenged chickens would suggest that

tissue uptake might have been affected (Hazelwood, 2000). Insulin is a powerful anabolic

hormone, and according to Hazelwood (2000) on a unit/unit basis even more potent than

mammalian insulin. These observations lead one to conclude that reovirus challenge might

have had a negative metabolic influence on infected chickens. Low concentrations of plasma

in insulin are correlated with body weight depression such as that shown by ARVCU98- and

S1733-challenged chickens. Doerfler et al. (2000), who worked with PEMS infected poults,

reported lower level of plasma insulin and lower body weight gain in PEMS-affected poults.

Sterner et al.(1988) used S1733-challenged chickens, which exhibited high plasma total

protein and plasma albumin suggesting a dysfunctional liver. Those observations coupled

with observations in the current investigation suggest that in ovo reovirus challenge resulted

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in hepatic dysfunction possibly caused by decreased glucagon and insulin in virus-challenged

chickens. The post hatch plasma IGF-I and IGF-II concentrations were not affected

significantly by the in ovo reovirus challenges. IGF-II expression was reported to be greater

than IGF-I in both chickens and turkeys (McMurtry, 1998), but in this investigation

comparable levels of both were found. The fact that neither IFG-I nor IGF-II was affected by

reovirus challenge in chickens was in contrast with observations reported by Doerfler et al.

(2000) who used PEMS-infected poults.

The plasma concentrations of the thyroid hormones in the in ovo reovirus-challenged

chickens was not affected significantly, which was in contrast to the depressions in T4 and T3

found in PEMS infected poults (Edens and Doefler, 1997a,b; Doefler et al., 2000). The T3/T4

ratio in the reovirus-challenged chickens was not significantly lower than the controls, but

the numerical decrease in conversion of T4 to T3 supporting the hypothesis of a metabolic

disorder in reovirus-challenged chickens.

Body weights were depressed as the result of virus challenge, but the virus-

challenged chickens were not fat, but they were well fleshed. Thus, the lack of an IGF-I and

IGF-II response would suggest that other factors might have been affected by the virus

challenge. Decuypere et al. (1987) has suggested that lean birds have a low plasma insulin

concentration but high plasma growth hormone concentration coupled with a low plasma T3

concentration. In this investigation, plasma T3 concentration in the control chickens was

slightly, but not significantly higher compared to the plasma T3 concentration in ARVCU98

and S1733 (1:100) reovirus-challenged groups. The plasma T4 concentrations in all the

virus-challenged broiler chickens were generally, but not significantly, higher as compared

with controls. Doerfler et al. (1998) reported that turkey poults with a PEMS infection had

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lower T4 to T3 conversion via deiodinases located in the liver, and this was demonstrated as a

significant decrease in the T3/T4 ratio. The results obtained in this investigation suggest that

there is a metabolic disorder associated with reovirus infection that appears to have been

overlooked in the past.

Alterations in enzymes such as ALT and AST in PEMS-infected poults were

consistent with the findings of Edens and Doerfler (1997) and Heggen et al., (2001). At 1 d

of age, the ALT levels of the virus-challenged groups were significantly elevated suggesting

a severe virus-related infective state in the liver. Elevated ALT activity was evident at 14 d of

age in S1733 challenged chickens, but by 28 d of age there was no virus effect. AST

activities were not affected significantly by virus challenge. These results were in contrast to

observations made in PEMS-challenged poults. It should be noted that ALT is more

indicative of liver damage than the AST, since the latter can be produced in tissues other than

the liver (Chang et al., 1994 ). Sterner et al., (1988) also reported no difference in the AST

activity between the negative control and S1733-challenged chickens. Elevated ALT levels

were expected as liver pathology is attributed to reovirus infection.

Tissue sections of thymus and bursa from control, ARVCU98, and S1733 in ovo

reovirus-challenged chickens were tested for the presence of viral antigen. At 14 and 21 days

of age there was no viral antigens detected. It is possible that by these ages the chickens were

recovering from the viremia. Heggen-Peay (2000) detected viral antigens in thymus and

bursa of Fabricius at 2, 5, 7 days post inoculation. Therefore, viral antigen detection might

be limited to a time of maximum virus expression at an earlier age.

A summary of the histopathology at 14 d post hatch revealed that eosinophilia-like

changes were enhanced in ARVCU98 and S1733 reovirus-challenged chickens. Edens et al.

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(1997c) have reported that the accumulation of eosinophils in lymphoid tissues such as the

bursa of fabricius and the thymus precedes the eventual degeneration (decreased weight loss

and decreased cellularity) of the tissue. By 28 d of age, loss of cellularity was detected in the

thymus of S1733 and ARVCU98 reovirus-challenged chickens. The degenerative responses

of the thymus of virus-challenged birds were also accompanied by increased numbers of

macrophages in that tissue. These results suggest that virus-challenged chickens try to mount

an immune response, but it is compromised during active infection leading to

immunodysfunction. These observations were consistent with the results reported by Heggen

et al., (1998).

When transmission electron micrographs of the pancreas, anterior pituitary, and

thyroid glands from reovirus-challenged chickens were examined, they revealed high

incidence of mitochondrial disruption similar to the findings liver cells from PEMS infected

poults (Doerfler and Edens, 1997). Mitochondria are the cellular sites for oxidative

metabolism. In the case of the virus-challenged chickens, this mechanism can become

uncoupled and metabolic disruption can become manifest. The TEMS show mitochondrial

abnormalities in thyroid, pancreas and pituitary of reovirus-challenged chickens. Thus, even

though plasma glucose levels of virus-challenged chickens were not significantly different

from the control, the ability of the virus-challenged chickens to utilize the primary energy

source will be impaired and the birds will not be capable of maintaining body growth

comparable with controls.

The TEMs show S1733 virus particles in the liver, which is believed to be the target

organ in avian reovirus infection (Jones, 1989; Kibenge, 1985; Mandelli, 1978), and

additionally ARVCU98 was found to be in the pancreas of ion ovo challenged chickens.

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Body feathering of ARVCU98 and S1733 reovirus-challenged chickens was less than

in control birds. Rosenberger et al., (1988) observed poor feathering in S1733-challenged

broilers, and it has been observed in broilers suffering from runting and stunting syndrome, a

condition linked to reovirus infection, poor feathering has been observed (Elmubarak et al.,

1990).

Thus, it was concluded that reovirus infection from both the turkey isolate and the

chicken isolate induce pathological signs that were comparable in chickens that were

challenged in ovo. The results of the reovirus infection caused a metabolic disorder that was

apparently of short duration but with extensive consequences. The metabolic hormones

appear to have been compromised and several endocrine glands were also altered even down

to ultrastructural level. The results from this investigation suggest that the ARVCU98, a

turkey isolate, can cause pathogenic changes in chickens as well as in turkey poults. It is

suggested without proof that ARVCU98 might have been a virus that might have been

involved in the index case of PEMS.

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Table 3.1: Percent hatchability and mortality of fertile eggs challenged with reovirus isolates. Treatment GroupA Control ARVCU98 S1733(1:100) S1733(1:500) Hatchability (%) 70 24 59 67

Viability at day 18 (%) 53 37 42 52

A Eggs were challenged via the yolk sac at day 9 of incubation, 100ul/egg

Table 3.2. Body weights of broiler chicks challenged with reovirus isolates at day 9 of incubation , Trial 1.

Body weights (g) (LSMeans+SE) A

TreatmentB 1 7 14 28

Control 37.32+0.55a 157+3.72a 400+6.9a 960+31a

ARVCU98 37.00+1.05a 14310.23ab 308+8.92b 869+76a

S1733 (1:100) 36.94+0.63a 135+4.30b 345+10.22b 853+76a

S1733 (1:500) 37.50+0.57a 130+3.96b 325+9.38b 824+29a

Pvalue 0.9169 <0.0001 <0.0001 0.1855

AMean body weights, at 1,7,14 and 28 days of age BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, unlike superscript differs significantly (P<0.05)

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Table 3.3. Body weights of broiler chicks challenged with reovirus isolates at day 9 of incubation, Trial 2. Body weights (g) (LSMeans+SE) A

TreatmentB 1 7 14 28 Control 44.2+0.68b 229.1+5.12a 418.1+11a 1149.5+33

ARVCU98 45.4+0.79b 165.6+6.1c 328+8.92c 1022.7+35

S1733(1:100) 45.7+068b 200.9+4.1b 382.3+13b 1092.2+33

S1733(1:500) 48.5+0.68a 189.7+5.1b 365.5+11b 1068.2+33

Pvalue 0.0004 <0.0001 <0.0001 0.0899 AMean body weights, at 1,7,14 and 28 days of age BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, unlike superscript differs significantly (P<0.05)

Table 3.4 Overall Mean Growth indexA of broiler chicks from 1-28 days of age. Treatment Growth IndexA Control 25.89 +0.52a

ARVCU98 23.72 +0.52b

S1733 (1:100) 23.48 +0.52b

S1733 (1:500) 22.75 +0.52b

Pvalue 0.0478

A Calculated by division of body weight at termination by bodyweight at day 1 of age (d28/d1)

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Table 3.5. Feed conversion ratio of broilers challenged with reovirus isolates at day 9 of incubation1. (Trial 1 and 2 combined.) Treatment 0-1week 0-2 weeks 0-4weeks Control 1.11+0.08a 1.11+0.05a 1.49+0.29a

ARVCU98 1.19+0.08a 1.33+0.05a 1.61+0.29a

S1733 (1:100) 1.05+0.08a 1.20+0.05a 1.48+0.29a

S1733 (1:500) 1.21+0.08a 1.29+0.05a 1.56+0.29a

Pvalue 0.5641 0.0985 0.9881 1Broiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. Table 3.6A. Effects on relative lymphoid organ weights of broiler chicks challenged in ovo reovirus isolates at day 9 of incubation. 14 days of age. Trial 1. Relative lymphoid organ weights (%) (LSMeans+SE) TreatmentB Bursa Thymus Spleen Liver Control 0.182+0.03b 0.243+5.1a 0.065+0.11a 4.09+0.33a

ARVCU98 0.252+0.03ab 0.155+6.1b 0.069+0.11a 5.66+0.35a

S1733(1:100) 0.215+0.03b 0.189+4.1ab 0.070+0.13a 3.56+0.33b

S1733(1:500) 0.277+0.03a 0.234+5.1a 0.089+0.11a 3.69+0.33b

Pvalue 0.0725 0.0674 0.0827 <0.0001 BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, means with unlike superscripts differ significantly (P<0.05)

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Table 3.6B. Effects on relative lymphoid organ weights of broiler chicks challenged in ovo reovirus isolates at day 9 of incubation. 28 days of age. Trial 1. Relative lymphoid organ weights (%) (LSMeans+SE) TreatmentB Bursa Thymus Spleen Liver Control 0.336+0.03b 0.289+0.03a 0.065+0.01a 3.23+0.13a

ARVCU98 0.247+0.03ab 0.133+0.07a 0.093+0.01a 3.21+0.28ab

S1733(1:100) 0.212+0.02b 0.233+0.03a 0.090+0.01a 2.68+0.16b

S1733(1:500) 0.262+0.03ab 0.189+0.05a 0.079+0.01a 3.12+0.13ab

Pvalue 0.0608 0.01634 0.4279 0.0452 BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, unlike superscript differs significantly (P<0.05)

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Table 3.7A. Effects on relative lymphoid organ weights of broiler chicks challenged in ovo reovirus isolates at day 9 of incubation. 14 days of age. Trial 2. Relative lymphoid organ weights (%) (LSMeans+SE) TreatmentB Bursa Thymus Spleen Liver Control 0.205 +0.02a 0.175+0.02a 0.064+0.01ab 3.971+0.27ab

ARVCU98 0.203+0.02a 0.171+0.02a 0.089+ 0.01a 4.751+0.30a

S1733(1:100) 0.196+0.02a 0.200+0.02a 0.080+0.01ab 3.888+0.30ab

S1733(1:500) 0.259+0.02a 0.201+0.02a 0.077+0.01ab 3.761+0.27ab

Pvalue 0.1555 0.7119 0.1418 0.1003 BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, means with unlike superscripts differs significantly (P<0.05)

Table 3.7B. Effects on relative lymphoid organ weights of broiler chicks challenged in ovo reovirus isolates at day 9 of incubation.28 days of age. Trial 2. Relative lymphoid organ weights (%) (LSMeans+SE) TreatmentB Bursa Thymus Spleen Liver Control 0.237+0.03a 0.198+0.02a 0.092+0.01a 3.400+0.19a

ARVCU98 0.262+0.03a 0.186+0.02a 0.088 +0.01a 3.429+0.19a

S1733(1:100) 0.248+0.02a 0.140+0.02a 0.084 +0.01a 3.171+0.21a

S1733(1:500) 0.266+0.02a 0.160+0.02a 0.087 +0.01a 3.261+0.17a

Pvalue 0.7145 0.1881 0.9479 0.7698 BBroiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. a,bIn a column, unlike superscript differs significantly (P<0.05)

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Table 3.8 Feather Scores of broilers challenged with reovirus isolates or PBS at day 9 of incubation1. (At 2 and 4 weeks of age.) Treatment Feather Score Control 5 ARVCU98 3 S1733 3 1Broiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation. Table 3.9 Necropsy observation of broilers inoculated of broilers challenged with reovirus isolates or PBS at day 9 of incubation1

Tissue Control ARVCU98 S1733 Intestines Unaffected Thin-walled Thin-walled gaseous gaseous 16/16 16/16 1Broiler chicks were challenged with ARVCU98, S1733 (1:100 and 1:500) and or PBS at day 9 of incubation.

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Table 3.10A. Histopathology of thymus from broiler chickens treated in ovo with reovirus.* Trial 1. Parameters Control ARVCU98 S1733 (1:100) S1733 (1:500) “Eosinophilia” (14d) + +++ ++ ++ Cell Loss (28d) U - -- -- Macrophage (28d) U ++ + + Table 3.10B. Histopathology of bursa from broiler chickens treated in ovo with reovirus.* Trial 2. Parameters Control ARVCU98 S1733 (1:100) S1733 (1:500) “Eosinophilia” (14d) U +++ ++ ++ Cell Loss (28d) U -- - - Macrophage (28d) U +++ + + * U-unaffected + Occasional increase ++Obvious increase +++Greatly increased - Some loss of cells from medullary portion of follicle -- Obvious loss of cells from medullary portion of follicle

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Table 3.11. Overall mean plasma profile of broiler chicks challenged with reovirus isolates at day 9 of incubation.

Plasma Control ARVCU98 S1733 S1733 Pvalue chemistry (1:100) (1:500) Glucose 376+ 14 a 337+ 14 a 382+ 15 a 377+14a 0.1079 (mg/dl) Glucagon 216+45a 120+8b 128+16b 147+14ab 0.0502 (pg/ml) Insulin 3597+212a 1938+92b 2363+192b 3731+529a 0.0077 (pg/ml) IGF-1 37+1.6 a 42+2.2 a 40+2.16 a 41+2.4 a 0.4562 (ng/ml) IGF-2 43+2.9 a 47+3.2 a 51+6.2 a 44+4.1 a 0.6424 (ng/ml) T3 1.76+0.14 a 1.60+0.16 a 1.64+0.109 a 1.79+0.62 a 0.6577 (ng/l) T4 8.54+0.69 a 9.04+0.46 a 8.63+0.74 a 9.3+0.77 a 0.8153 (ng/ml) T3/T4 ratio 0.207+.04 a 0.177+ 0.03a 0.210+ .03 a 0.204+0.03 a 0.7691

a,b In a row, means with different superscripts differ significantly (P<0.05)

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Table 3.12. Liver function assay (mg/Ul) of broiler chicks challenged with reovirus isolates1 at day 9 of incubation overtime Liver function assay (mg/Ul) (LSMeans+SE) ALT AST Treatment Day 1 Day 14 Day 28 Day 1 Day 14 Day 28 Control 111+19 b 32 + 11b 59+ 22a 206+25 a 73+ 16 a 233+31 a ARVCU98 178 +26a 28 + 11b 39 + 27a 264+ 26 a 88+21 a 236+43 a S1733 177 +22a 64 + 8a 54 + 22a 244+19 a 84+12 a 230+27 a Pvalue 0.0562 0.0291 0.8465 0.2670 0.8122 0.9908 1 Broilers were inoculated with either ARVCU98, S1733 or control PBS ab In a column, unlike superscript differs significantly P<0.05

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Science

Control

ReovirusS1733

Two weeks

Figure 3.1. Comparison of wing feathers of control and S1733 challenged broiler chickens

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Science

ControlFour Weeks

Figure 3.2. Control broiler at four weeks of age.

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Science

S1733 ReovirusFour Weeks

Figure 3.3. S1733 challenged chicken at four weeks of age.

88

Day 14 Control Thym us 400X

Figure 3.4. Control thymus at 14 days of age. Reticular structures (RS) are the Pale areas throughout the medullary (M) regions. The darker staining area is the cortex (C ). 400X, Light Microscopy.

RS

C

M

89

14 Day ARVCU98 Thymus 400X

Figure 3.5. ARVCU98 challenged, thymus at 14 days of age. Reticular structures (RS) are the pale areas throughout the medullary (M) regions. The darker staining area is the cortex (C). 400X, Light Microscopy.

PE

E

RS

M

C

90

14 Day S1733 Thymus 400X

Figure 3.6. S1733 challenged thymus at 14 days of age. Reticular structures (RS) are the pale areas throughout the medullary (M) regions. The darker staining area is the cortex (C). 400X, Light Microscopy.

C M RS

91

28 Days

Figure 3.7. Control thymus at 28 days of age. Reticular structures are the pale areas throughout the medullary ( M) regions. The darker staining area is the cortex ( C ). 400X, Light Microscopy.

C

M

92

28 Days

Figure 3.8. ARVCU98 challenged thymus at 28 days of age. Reticular structures are the pale areas throughout the medullary (M) regions. The darker staining area is the cortex (C ). 400X, Light Microscopy.

C M

RS

93

28 D ays

Figure 3.9. S1733 challenged thymus at 28 days of age. Reticular structures are the pale areas throughout the medullary (M) regions. The darker staining area is the cortex (C). 400X, Light Microscopy.

C

M

94

14 Days Control Bursa 400X

Figure 3.10. Control bursa of Fabricius at 14 days of age. LM is lamina propria, M is medulla; C is cortex.

LM

M

C

95

14 Days ARVCU98 Bursa 400X

Figure 3.11A. ARVCU98 challenged bursa of Fabricius at 14 days of age. LM is lamina propria; M is medulla; C is cortex; E is “eosinophilia”.

E

C

M

LM

96

14 Days ARVCU98 Bursa

Figure 3.11B. ARVCU98 challenged bursa of Fabricius at 14 days of age. M is medulla; C is cortex.

C

M

97

S1733 Bursa 400X

Figure 3.12. S1733 challenged bursa of Fabricius at 14 days of age. M is medulla; C is cortex. E is “eosinophilia”. 400X. Light microscopy.

E

M

C

E

98

28 Days

Figure 3.13. Control bursa of Fabricius at 28 days of age. F is follicle; C is cortex; M is medulla; UC is undifferentiated epithelial cells; PE is pseudostratified epithelium. 200X. Light microscopy.

F C

M

PE

UC

99

Figure 3.14. ARVCU98 bursa of Fabricius at 28 days of age. F is follicle; C is cortex; M is medulla; UC is undifferentiated epithelial cells; 200X. Light microscopy.

C

M

LM UC

100

28 Days

Figure 3.15. S1733 challenged bursa of Fabricius at 28 days of age. C is cortex; M is medulla; UC is undifferentiated epithelial cells;. 200X. Light microscopy.

M

C

UC

LM

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Control Pancreas 5K

ZG

RER

ZG

N

COR

Figure 3.16. This is a pancreatic acini cell from a control broiler chicken pancreas. The acini cell is distinguished from beta cell by the anatomy of the zymogen granule which is not bound by membrane in the acini cell. 5000X magnification.

N is nucleus COR is cisternae of reticulum-develops as concentric and almost parallel waves of RER around nucleus after feeding RER is rough endoplasmic reticulum M is mitochondria ZG is zymogen granule

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ARVCU98 Pancreas 5k

M

RER

ZG

N

N

MRER

COR

Figure 3.17. This is a pancreatic acini cell from an ARVCU98 reovirus-challenged broiler chicken pancreas. The acini cell is distinguished from beta cell by the anatomy of the zymogen granule which is not bound by membrane in the acini cell. 5000X magnification.

N is nucleus COR is cisternae of reticulum-develops as concentric and almost parallel waves of RER around nucleus after feeding RER is rough endoplasmic reticulum M is mitochondria ZG is zymogen granule

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S1733 Pancreas 5K

N

ZGM

M

N N

N

RER

ZG

RER

M

MM

ZG

Figure 3.18. This is a pancreatic acini cell from a S1733 reovirus-challenged broiler chicken pancreas. The acini cell is distinguished from beta cell by the anatomy of the zymogen granule which is not bound by membrane in the acini cell. 5000X magnification.

N is nucleus COR is cisternae of reticulum-develops as concentric and almost parallel waves of RER around nucleus after feeding RER is rough endoplasmic reticulum M is mitochondria ZG is zymogen granule

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Control Anterior Pituitary 5k

N

N M

SG

N

M

SG

CB

CL

Figure 3.19. These are chromophobe and chromophil cells in the anterior pituitary of a control broiler chicken. 5000X magnification.

CB-is chromophobe CL is chromophil N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule

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ARVCU98 Anterior Pituitary 5K

N

CL CB

NSG

SG

M

SG

RER

RER

CL

Figure 3.20. These are chromophobe and chromophil cells in the anterior pituitary of an ARVCU98 reovirus-challenged broiler chicken. 5000X magnification.

CB-is chromophobe CL is chromophil N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule

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S1733 Anterior Pituitary 5k

CB

CBSG

SG

MM

RERM

N

N

M

RER

CL

CB

Figure 3.21. These are chromophobe and chromophil cells in the anterior pituitary of a S1733 reovirus-challenged broiler chicken. 5000X magnification.

CB-is chromophobe CL is chromophil N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule

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Control Thyroid 5K

N

SGM

RER

RER

MCOR

BC

BC

Figure 3.22. These are follicular epithelial cells in the thyroid gland from a control broiler chicken. 5000X magnification.

COR is cisternae of the reticulum N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule

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ARVCU98 Thyroid 5K

N

N

N

M+RER

M

M+RER

COR

BC

BC

Figure 3.23. These are follicular epithelial cells in the thyroid gland from an ARVCU98 reovirus-challenged broiler chicken. 5000X magnification.

COR is cisternae of the reticulum N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule BC is blood cell in a capillary

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S1733 Thyroid 5K

M+RER

N

M+RER

COR

G

Figure 3.24. These are follicular epithelial cells in the thyroid gland from a S1733 reovirus-challenged broiler chicken. 5000X magnification.

COR is cisternae of the reticulum G is Golgi N is nucleus RER is rough endoplasmic reticulum M is mitochondria SG is secretory granule BC is blood cell in a capillary

BC

110

Figure 3.25. Reovirus particle in the liver of a S1733 reovirus-challenged broiler chicken. 70,000X magnification; bar represents 200nm.

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Figure 3.26. ARVCU98 reovirus being shed from pancreatic cells from a broiler chicken. Magnification is 20,000X.

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Figure 3.27. Electron micrographs comparing ARVCU98 reovirus isolated from turkey poults and S1733 reovirus isolated from chickens. Magnification : 89,600X.

ARVCU98

S1733

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CHAPTER 4

Efficacy of Hyperimmunizing Breeder Hens to Protect Progeny Against PEMS-Associated ARVCU98 Reovirus Challenge

ABSTRACT A PEMS-associated ARVCU98 vaccine (105.7 TCID50; 1mL/hen) in Freund’s

adjuvant was injected subcutaneously on the back of the neck at the initiation of lay (30

weeks of age) into 8 breeder turkey hens, and these were called vaccinated hens. Another 8

hens were control injected with LMH cell supernatant. Booster vaccinations were given two

times separated by intervals of 2 weeks. Egg collection was done for a period of two week

intervals at 30, 45 and 60 days after booster vaccinations (DAB). At hatch, poults from the

two parents stocks were challenged orally with PEMS-associated ARVCU98 reovirus

(200ul/poult) and were designated as 1) vaccinated and challenged 2) vaccinated but not

challenged 3) unvaccinated and challenged 4) unvaccinated but not challenged. At 6 days

after homologous reovirus challenge, body weights of challenged poults in the 45 DAB

group was depressed but not in the 30 and 60 DAB groups. At 10 days after reovirus

challenge, body weights of reovirus-challenged poults in the 30 DAB group were decreased

but this was not observed in poults from the 45 and 60 DAB groups. At both 6 and 10 days

after reovirus challenge, bursa of Fabricius relative weights were decreased significantly in

reovirus-challenged poults from both control and vaccinated breeders. Lymphoid organ

relative weights of poults in the 45 DAB group were decreased after reovirus-challenge in

both vaccinated and control parents stock. Thymus relative weights were decreased by

reovirus challenge in poults from vaccinated control and breeder hens at 10 days after

reovirus challenge. Spleen relative weights in poults from control and vaccinated breeders

were increased significantly at 6 and 10 days after reovirus challenge in the 45 and 60 DAG

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groups, respectively. At 6 days after reovirus challenge, liver relative weights in reovirus-

challenged poults from control and vaccinated breeders were decreased significantly in the

60 DAB group. At 10 days after reovirus challenge, liver relative weights in reovirus-

challenged poults from control and vaccinated breeders were decreased significantly in the

30 DAB group, but in the 45 DAB group, liver relative weights were increased significantly

in reovirus-challenged poults from control and vaccinated breeder hens. These observations

suggest that vaccination for ARVCU98 only had transitory effects in the progeny of breeder

turkey hens subjected to reovirus challenge at hatch.

Key Words: poult enteritis and mortality syndrome, reovirus, vaccination

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INTRODUCTION

The ARVCU98 reovirus has been implicated as one of the etiologic agents that cause

PEMS infection in turkey poults (Heggen-Peay et al., 2001; Chapters 2 and 3). PEMS has

been characterized as a transmissible enteric disease of young turkey poults with high rates of

morbidity and mortality and has been classified as a disease with multifactorial etiology.

PEMS infection results in reduced weight gain, lymphoid organ and liver atrophy, and

immunosuppression/immunodysfunction. The immunodysfunction was characterized by

enhanced proinflammatory cytokine production by activated macrophages and by intestinal

inflammation, abnormal gut motility and anorexia (Heggen-Peay et al., 2000). Although

poults may survive the infection, their body weights remained below market standards with

no signs of compensatory weight gain after infection.

Since reovirus infection is age-related, efforts to control its spread have focused on

hyperimmunizing breeder hens (Meanger et al., 1997). Immunity against certain infections

can be achieved in two ways 1) vaccination or infection (active immunity) or 2) by transfer

of antibodies or lymphocytes from actively immunized animals (Abbas et al., 2001). The

transfer of passive antibodies from dam to progeny via the egg provides temporary resistance

to infectious agents by providing immediate protection. Innate/passive immunity is time

dependent as it declines to ineffectual levels by 10-20 days after hatch (Tizard, 1992).

Innate/passive immunity results from transfer of maternal IgG to the developing yolk that

ultimately transfers to the embryonic circulation (Saif et al., 1993).

Thus, it was hypothesized that vaccination of turkey breeder hens with ARVCU98

attenuated vaccine just before initiation of lay would confer immunity to progeny and protect

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them against homologous reovirus challenge at day of hatch. The results of this investigation

on the pathogenicity of ARVCU98 are reported in this paper.

MATERIALS AND METHODS

Animal Care. This project was approved and conducted under the supervision of the North

Carolina State University Animal Care and Use Committee which has adopted Animal Care

and Use Guidelines governing all animal use in experimental procedures.

Animal husbandry. This investigation was conducted with three separate hatches of poults.

Two turkey breeder hen groups of eight hens per group were designated as control or

vaccinated/hyperimmunized. These hens were sourced from the North Carolina State

University Flock (Nicholas strain). The turkey breeder hens were 30 weeks old when they

were vaccinated (1ml/hen; subcutaneously at back of the neck) with the inactivated PEMS-

associated ARVCU98 reovirus (105.7 TCID50) in incomplete Fruend’s adjuvant, and a booster

vaccination was administered after two weeks . Eggs were collected at two weeks interval

commencing at 30, 45 and 60 days after the booster vaccination. Eggs were incubated under

standard conditions for turkey eggs. At hatch, poults from the two breeder groups were wing-

banded, weighed, challenged orally with homologous ARVCU98 reovirus (100ul/poult) and

placed into plastic isolators (Standard Safety, Chicago, IL). There were four groups that were

designated as 1) vaccinated and challenged 2) vaccinated but not challenged 3) unvaccinated

and challenged 4) unvaccinated but not challenged. Poults in these four groups were placed

in separate isolators. A total 20 poults per isolator were of started in an ambient temperature

of 35ºC that was decreased by 4ºC at intervals of 7 days. There was continuous incandescent

lighting provided in this investigation. Feed and water were provided for free access. A

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special irradiated feed ,sterilized Mazuri feed, (Purina Mills, St. Louis,MO), equivalent to the

standard North Carolina Agriculture Research Service turkey starter diet, was used to assure

that no extraneous bacterium or virus was unknowingly involved in the investigation. Feed

and water for the poults in this investigation were placed in bulk into the isolators to prevent

accidental loss of biosecurity, and scales for determination of body weight were also held in

the isolators until the end of the investigation. Poults removed from the isolators were first

caught using the glove ports in the isolators, placed into an antechamber that was opened and

resealed before a second seal was removed from the isolator to allow collection of the sample

poults. After the sample poults had been removed from the antechamber, it was resealed and

fumigated with a chlorine compound.

Feed and water were given ad libitum. Body weight and lymphoid organ weights

were measured to assess the effect of dam vaccination and post hatch ARVCU98 reovirus

challenge.

Vaccine production. LMH cells were infected with ARVCU98 (10 5.7 TCID50), frozen and

thawed 3x and clarified by low speed centrifugation. Formaldehyde was added to inactivate

the virus with a final concentration of 0.1%. Equal volume of incomplete Freund’s adjuvant

was added to the mixture.

Body weights and lymphoid organ weights. Body weights were taken at day of hatch, and

days 6 and 10 post hatch. Bursa of Fabricius, thymus (left side of the neck), spleen and liver

were removed and weighed from 8 birds per treatment group. Lymphoid organs were

expressed as percentage of body weight and reported as organ relative weights.

Statistical Analysis. The data from the completely randomized experimental design were

subjected to analysis of variance testing using the General Linear Models procedures of SAS

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(SAS Institute, 1999). If the main effects of treatment or time were significant, treatment

means were separated using the least significant difference procedure of SAS (1999).

Significance was set at P≤ 0.05.

RESULTS

In vivo studies. Tables 4.1 and 4.2 present the treatment mean body weights of control and

reovirus-challenged poults from control and hyperimmunized turkey breeder hens. At 6 days

after homologous ARVCU98 challenge, body weights of reovirus-challenged poults in the 45

DAB group was depressed but not in the 30 and 60 DAB groups (Table 4.1) . At 10 days

after ARVCU98 challenge, body weights of ARVCU98-challenged poults in the 30 DAB

group were decreased but this was not observed in poults from the 45 and 60 DAB groups

(Table 4.2). At both 6 and 10 days after ARVCU98 challenge, bursa of Fabricius relative

weights were decreased significantly in ARVCU98-challenged poults in the 45 and 60 DAB

groups from both control and vaccinated breeders (Tables 4.3 and 4.4). Thymus relative

weights at 6 and 10 days after ARVC98 challenge are presented in Table 4.5 and 4.6. At 10

days after ARVCU98 challenge, thymus relative weights of poults in the 45 DAB groups

were decreased significantly in ARVCU98-challenged poults from both control and

vaccinated breeders (Table 4.6). Spleen relative weights in ARVCU98-challenged poults

from control and vaccinated breeders were increased significantly at 6 and 10 days after

ARVCU98 challenge in the 45 and 60 DAB groups, respectively (Table 4.7 and 4.8). At 6

days after ARVCU98 challenge, liver relative weights in virus-challenged poults from

control and vaccinated breeders were decreased significantly in the 60 DAB group (Table

4.9). At 10 days after virus challenge, liver relative weights in virus-challenged poults from

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control and vaccinated breeders were decreased significantly in the 30 DAB group, but in the

45 DAB group, liver relative weights were increased significantly in virus-challenged poults

from control and vaccinated breeder hens (Table 4.10).

DISCUSSION

The definitive etiologic agents that cause PEMS still elude researchers. Even though, several

putative etiologic agents have been implicated in PEMS, research to identify veterinary

procedures to prevent the disease are not available. The PEMS disease was very expensive

and more than $161 million dollars in revenue were lost by the turkey industry in the

Southeastern United States through 1997. In lieu of a vaccination program, the turkey

industry initiated a very strict biosecurity program. The biosecurity program was helpful in

breaking the disease cycle, but it is a process that can be easily by-passed.

One way to possibly deter PEMS infection is through a vaccination program that

would immunize the poults against one or all of the potential etioloic agents implicated in the

disease. The turkey reovirus ARVCU98 has been implicated as a virus that might

compromise the intestinal tract of poults and set up the conditions leading to fulminating

PEMS (Qureshi et al., 1997, Heggen-Peay et al., 2000). Post-hatch vaccination would, by

necessity, have to be administered to newly hatched poults. However, the potential exists for

maternal antibody interference against successful vaccination of newly hatched poults

because maternal antibody persists in chicks and poults for 10-20 days post hatch (Tizard,

1992). Therefore, use of an inactivated ARVCU98 reovirus vaccine in an incomplete

Freund’s adjuvant in turkey breeders before the initiation of lay was believed to be a means

to increase protection against the ARVCU98 reovirus in poults from the vaccinated breeders.

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The results were not as clear as one would hope, but there were indications that the

vaccination program might have possibilities. Body weights of reovirus-challenged poults

from control and vaccinated breeders were reduced minimally at 6 days after reovirus

challenge in the 45 DAB group and at 10 days in the 30 DAB group. Furthermore, the

thymus weight was reduced only at 10 days after reovirus challenge in the 45 DAB group of

PEMS-challenged poults from control and vaccinated breeders. Thymus weight decrease is

one of the primary signs of reovirus in poults (Edens et al., 1997abc; Qureshi et al., 1997,

Heggen-Peay et al., 2000). The observation that relative weights of thymus were decreased

by reovirus challenge only one time in this investigation was very encouraging for the

possible control by PEMS-associated reovirus vaccination. However, the route of

administration of the pilot vaccine used in this investigation was via subcutaneous injection

into the breeder hens. Van der Heide (1976), working with chickens, demonstrated that

maternal immunization protected post hatch chicks from experimental reovirus challenge

only when the vaccine was administered to the dam via the oral route. Nevertheless, the

results from this investigation still strongly suggest that vaccination against reovirus in the

breeder hen holds great potential, and it is suggested that additional work should be

conducted to look at alternative means of vaccine administration that would generate the best

transfer of maternal antibody to protect the poult from PEMS-associated reovirus or other

viruses associated with PEMS.

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Table 4.1. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on body weight of progeny at 6 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue 30 DAB 65.85 70.00 66.12 63.25 0.3008

+ 2.62a + 2.45 a + 2.45a + 2.46a

45 DAB 90.50a 81.33b 84.71b 95.37a 0.0494

+3.68 +3.47 +3.93 +3.67 60 DAB 90.62 90.29 93.0 86.87 0.1898

+1.98a +2.1a +1.9a +1.98a

ab, in a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.2. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on body weight of progeny at 10 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 113.75 89.25 84.00 114.00 <0.0001

+ 3.74a + 3.74b + 3.74b +3.74a

45 DAB 136.8 120.25 133.25 139.78 0.0737 +6.74 +5.33 +5.33 +5.01

60 DAB 149.11 145.25 140.89 153.62 0.2323 +4.29 +4.56 +4.30 +4.56

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.3. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on bursa weights (% of BW) of progeny at 6 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 0.094 0.094 0.090 0.082 0.5986

+ 0.01a + 0.01a + 0.01a + 0.01a

45 DAB 0.147 0.125 0.122 0.159 0.0474 +0.01 ab +0.01 ab +0.01 b +0.01 a

60 DAB 0.209 0.154 0.130 0.161 0.0243 +0.02 a +0.02 b +0.016 b +0.02 ab

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.4. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on bursa weights (% of BW) of progeny at 10 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 0.089 0.081 0.087 0.086 0.8517

+ 0.01a + 0.01a + 0.01a + 0.01a

45 DAB 0.150 0.118 0.101 0.143 0.0014 +0.10 a +0.01 b +0.01 b +0.01a

60 DAB 0.209 0.147 0.130 0.182 0.0041 +0.01a +0.01ab +0.01b +0.01a ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.5. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on thymus weights (% of BW) of progeny at 6 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 0.122 0.122 0.111 0.122 0.7732

+ 0.01a + 0.01a + 0.01a + 0.01a

45 DAB 0.104 0.110 0.093 0.097 0.7895 +0.11a +0.09a +0.01a +0.06a

60 DAB 0.120 0.108 0.115 0.120 0.9499 +0.15a +0.02a +0.15a +0.15a

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

126

Table 4.6. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on thymus weights (% of BW) of progeny at 10 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 0.137 0.118 0.101 0.132 0.2044

+ 0.01a + 0.01a + 0.01a + 0.01a

45 DAB 0.138 0.096 0.104 0.114 0.0218 +0.01a +0.01b +0.01b +0.01ab

60 PDB 0.134 0.110 0.108 0.113 0.1523 +0.01 a +0.01ab +0.01b +0.01ab

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.7. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on spleen weights (% of BW) of progeny at 6 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DBP 0.046 0.048 0.044 0.038 0.7732

+ 0.005a + 0.004a + 0.004a + 0.004a

45 DAB 0.055 0.086 0.079 0.057 0.0108 +0.04b +0.04a +0.04a +0.04b

60 DAB 0.050 0.075 0.066 0.101 0.7506 +0.03a +0.04a +0.03a +0.03a

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

128

Table 4.8 Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on spleen weights (% of BW) of progeny at 10 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 0.050 0.052 0.049 0.055 0.7877

+ 0.005a + 0.004a + 0.004a + 0.004a

45 DAB 0.054 0.143 0.079 0.057 0.3579 +0.01a +0.01a +0.01a +0.01a

60 DAB 0.057 0.083 0.069 0.054 0.0078 +0.01b +0.01a +0.01ab +0.01b

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.9. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on liver weights (% of BW) of progeny at 6 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 3.349 3.631 3.364 3.395 0.4569

+0.136 a +0.127 a +0.127 a +0.127 a

45 DAB 4.29 4.04 3.83 3.99 0.4699 +0.01a +0.01a +0.01a +0.01a

60 DAB 3.301 2.893 2.300 3.283 0.0036 +0.08 a +0.09 b +0.08 b +0.08a

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

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Table 4.10. Effect of hyperimmunization of turkey breeder hens against ARVCU98 reovirus on post hatch ARVCU98 challenge on liver weights (% of BW) of progeny at 10 days after reovirus challenge. Breeder Control Control Vaccinated Vaccinated Progeny Non-challenged Challenged Challenged Non-challenged Pvalue

30 DAB 4.176 3.329 3.071 4.139 0.0019

+0.22a +0.22b +0.22b +0.22a

45 DAB 3.413 4.215 4.437 3.194 <0.0001 +0.01b +0.01a +0.01a +0.01b

60 DAB 3.436 2.988 3.399 3.469 0.4178 +0.21a +0.23a +0.22a +0.23a

ab , In a row unlike superscript differs significantly (P<0.05) DAB- Day after boost

131

SUMMARY AND CONCLUSION

The purpose of this research was to further characterize PEMS in terms of

pathogenicity. In earlier studies on PEMS, poults were experimentally infected with either

direct contact with seeder poults or oral exposure to infected homogenates of either feces,

intestinal tracts, or thymus. Since no single entity has been directly linked to the disease

process, PEMS was classified as a multifactorial etiologic disease potentially caused by

several viruses and bacteria. Results from these early studies established signs of PEMS as

inhibition of weight gain, lymphoid organ (specifically thymus) degeneration, and liver

atrophy. Lymphoid organ pathology led to an immunodyfunctional state characterized by

several alterations in the immune system such as macrophage-induced cytokines and nitrite

production and changes in the lymphocytic and mononuclear phagocytic system.

The first objective of this investigation was to compare the pathogenicity of PEMS-

associated astrovirus (TastOSU, a turkey isolate), reovirus (ARVCU98, a turkey isolate), and

ARVCU98 in the presence or absence of atypical E. coli frequently isolated from PEMS-

infected poults. This combination agent investigation reported in Chapter 2 was conducted to

assess the effects of these organisms on poult body weight gain and lymphoid organ

integrity. The ARVCU98 reovirus depressed body weight gain between 10-17 DPI. The

presence of E. coli in the combination study did not result in significant differences

compared with the control, but the presence of the E. coli was consistently associated with

lower body weight gains. A decreased thymus relative weight in response to pathogen

challenge approached significance (P=0.07) in comparison with the control poults, and these

results confirmed earlier reports of thymic atrophy due to astrovirus infection (Koci et al.,

132

2000; Schultz-Cherry et al., 1999; Qureshi et al., 1999). Although the lymphoid cell

functions and population were not examined in this investigation, decreased organ relative

weight and decreased body weight gain were in the same order of magnitude as results

reported by Qureshi et al. (1999), wherein thymus degeneration was associated with lower

lymphocytes counts and reduced lymphoproliferative abilities in TastOSU-challenged poults.

Decreased immunologic status has been and is characteristic of PEMS infection. The

presence of E. coli in the combination of pathogens ( reovirus + astrovirus+ E. coli) caused

the lowest in weight gain and also had a negative influence on lymphoid organ relative

weights.

Based on these results, PEMS infection with either ARVCU98 or TastOSU or their

combination negatively influences body weight gain and lymphoid organ relative weights.

This condition may be exacerbated by the presence of atypical E. coli frequently isolated in

PEMS-susceptible poults. In poults exposed to these organisms in a natural setting,

fulminating PEMS can be induced when there is interaction of many other factors that are

common to the disease.

Vertical transmission of PEMS has been disputed ever since the disease was

identified as a problem for the turkey industry in North Carolina. Nevertheless, it has been

suggested that the possibility does exist. In Chapter 3, the second investigation in this thesis,

instead of direct contact exposure to induce PEMS, an attempt was made to use the in ovo

route as a means to infect embryos at 9 days of embryonation. Another innovative approach

in this second study was to use the chicken embryo as the experimental model to test both

turkey (ARVCU98 reovirus) and chicken (S1733 reovirus) isolates in ovo to evaluate the

potential of zoonosis as well as vertical transmission of the reoviruses. Body weight gain,

133

lymphoid organ integrity, liver pathology, and pathophysiological alterations in the hatched

chickens were assessed over a period of 28 days post hatch.

Body weight gains of chickens exposed in ovo to the chicken and turkey reovirus

were less than control chicken weight gains. This observation indicates that the ARVCU98

turkey isolate had the potential to be transmitted vertically in chicken embryos. From this

observation, it was inferred that the index case of PEMS might have resulted from cross

contamination of a chicken virus into turkey poults and from that point onward, the virus

may have mutated to become the causative agent that conditioned the intestinal tract to be

susceptible to the other putative virus and bacterial agents associated with PEMS. However,

the inferences are only limited to less weight gain because other signs of PEMS such as

degeneration of bursa, thymus, spleen and liver were not immediately evident, but, during

necropsy, it was noted that the virus infected broilers had thin-walled, gas-filled, frothy

intestines. This intestinal pathology was earlier associated with increased activity of

macrophage related inflammatory cytokines and nitrite production (Heggen-Peay, 2001). It

was subjectively noted that S1733 (both 1:100 and 1:500) infected broilers had heart and

kidney enlargement, which was absent in the ARVCU98 reovirus and control birds. The in

ovo virus challenges extended to decreased metabolic activity as indicated by decreased

feather growth and development. When scoring was done, control birds scored 5 (best) while

virus (ARVCU98 and S1733) infected chickens had a whole body feather score of 3.

More importantly, the results from this investigation showed that both the turkey and

chicken reovirus isolates had significant negative effects on metabolically active hormones.

Both glucagon and insulin were depressed in the virus challenged broiler chickens, but

plasma glucose levels were not affected by virus challenge. The IGF-I and IGF-II levels were

134

not significantly affected by in ovo virus challenge. There was a slight decrease in the T3/T4

ratio for the all the virus challenged broilers suggesting that the conversion of T4 to T3 had

been decreased. The hormonal imbalances found in this investigation were similar in the

result reported by Doerfler et al (2000). Thus, it can be implied that the decrease in over all

body weight gain in reovirus infected chickens/turkeys can be partially explained by

metabolic disturbance that interferes with, muscle and skeleton development. An

examination of transmission electron micrographs of the pancreas, anterior pituitary and

thyroid revealed mitochondrial damage (degeneration) as a primary response to reovirus

infection, but other cellular morphological changes also signaled virus-mediated disruption

of function. Condensation of rough endoplasmic reticulum was commonly observed in cell

from virus infected chickens, and this was associated with decreased production of secretory

granules in the cell cytoplasm. Additionally, in the pancreas, it appeared that reovirus

infection had inhibited zymogen granule secretion which would help to explain problems

associated with increased feed conversions in field cases of reovirus and PEMS infection in

the field. Mitochondrial degeneration in most of the tissues examined with transmission

electron microscopy must be considered as an indicator of generally decreased metabolic

activity, which would be consistent with the observation that muscle and organ wasting

exhibited by chickens with reovirus infection and turkeys with PEMS is due to decreased

mitochondria-based energy metabolism. The ARVCU98 was detected in the pancreas and

S1733 was easily detected in the liver of infected chickens in this investigation.

The histopathological effect of in ovo virus challenge on bursa and thymus was

evaluated, and between 14 and 28 days of age loss of cellularity eosinophilia was observed in

tissues from all the virus-challenged broilers. In the ARVCU98-infected broilers,

135

macrophages were observed frequently in the thymus. The loss of cellularity in both the

thymus and bursa of Fabricius was associated with accumulation of the eosinophil-like

granulocytes.

In chapter 4, vaccination/hyperimmunization of turkey breeder hens against the

reovirus ARVCU98 was done to assess its potential in preventing reovirus-mediated enteritis

in poults from the hyperimmunized breeders. It was believed that passive antibody transfer

to progeny of the ARVCU98 vaccinated breeders would be sufficient to prevent enteritis

resulting from ARVCU98 oral exposure of the poults on the day of hatch. Protection was

limited to day 6 post inoculation from the 30 days after boost treatment group progeny from

either vaccinated or non-vaccinated control breeders. Those progeny from the 45 and 60 days

after boost groups had sporadic signs of protection against the effects of exposure to reovirus

after hatch. The route of administration of the pilot vaccine used in this investigation was via

subcutaneous injection into the breeder hens. Van der Heide (1976), working with chickens,

demonstrated that maternal immunization protected post hatch chicks from experimental

reovirus challenge only when the vaccine was administered to the dam via the oral route.

Nevertheless, the results from this investigation suggest that vaccination against reovirus in

the turkey breeder hen holds great potential, and it is suggested that additional work be

conducted to look at alternative means of vaccine administration that would generate the best

transfer of maternal antibody to protect the poult from PEMS-associated reovirus or other

viruses associated with PEMS.

In conclusion, important findings in this research program were found. First, PEMS-

associated agents such as ARVCU98 and TastOSU cause depressed body weight gain and

promote lymphoid organ and liver atrophy. The presence of E. coli exacerbates the pathology

136

associated with the reovirus and astrovirus infections. These results can even be more severe

under field exposure where there are uncontrolled conditions that might facilitate more

severe disease. Second, egg transmission was shown to be a possibility with avian reoviruses

of both turkey and chicken origin. More importantly, this investigation showed that the

turkey reovirus isolate, ARVCU98, can infect broilers challenged in ovo and cause

significant pathology in the hatchling. There appeared to be parallel effects related to

ARVCU98 and S1733 infections although severity of their induced pathologies was

different. Reovirus challenges in ovo created pathological changes that resulted in conditions

that were likened to development of metabolic disease in the infected hatchlings. This

conclusion was drawn from observations on metabolically active plasma hormone

concentrations and ultrastructural changes in the endocrine glands in which these hormones

were secreted. Third, hyperimmunization of breeder turkey hens against the turkey reovirus

isolate, ARVCU98, provided limited protection to progeny, which were given an ARVCU98

challenge at hatch. An alternative breeder hen immunization route, oral vaccination, might be

more effective that subcutaneous inoculation. Additional work must be conducted to examine

many of the questions arising from this research.

137

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