VIRULENCE AND PATHOGENESIS OF NEWCASTLE DISEASE VIRUS ISOLATES FOR DOMESTIC CHICKENS

ISOLATES FOR DOMESTIC CHICKENS
GLAUCIA DENISE KOMMERS
(Under the Direction of Corrie C. Brown and Daniel J. King)
ABSTRACT
Newcastle disease virus (NDV) isolates vary greatly in virulence and pathogenicity depending on several factors including the host species and the infecting virus strain. This research was performed in order to investigate the effect of serial passages of several NDV isolates in domestic chickens. Six isolates were recovered from pigeons and six isolates had heterogeneous origin (recovered from chickens, wild, and exotic birds). A monoclonal antibody (MAb) panel revealed that four of the pigeon-origin isolates were the variant pigeon paramyxovirus-1 (PPMV-1) and two of them were avian paramyxovirus-1 (APMV-1) isolates. Pathotyping tests performed before and after passage in chickens demonstrated increased virulence of the passaged PPMV-1 isolates and high virulence of the original isolates of APMV-1. However, the PPMV-1 were still of moderate virulence for chickens after passages. The fusion protein cleavage site amino acid sequence of all six pigeon-origin (PPMV-1 and APMV-1) isolates was typical of virulent NDVs. Although the results of the pathotyping tests indicated a virulence increase of all passaged PPMV-1 isolates, clinical disease was limited to depression and nervous signs in some of the chickens inoculated intraconjunctivally. However, severe lesions were observed mostly affecting the heart and brain after intraconjunctival inoculation with passaged PPMV-1 isolates. Pigeons must be considered seriously as a potential source of NDV infection and disease for commercial poultry flocks. All six heterogeneous-origin isolates were characterized by reactivity to MAbs as members of the APMV-1 serotype. Three isolates showed low virulence for chickens before and after passage and had the F protein cleavage site typical of low virulence viruses. The other three heterogeneous-origin isolates were classified by the pathotyping tests and sequence analysis of the F protein cleavage site as moderate to highly virulent for chickens. An isolate recovered from an exotic dove had marked virulence increase after passage. The exact mechanism for the virulence increase observed with this isolate remains undefined. A pathogenesis study with the chicken-passaged isolates revealed that two of them were highly virulent for chickens, with marked tropism for lymphoid tissues. The results reported here demonstrate the high risk for domestic chickens represented by some NDV- infected non-poultry species.
INDEX WORDS: Apoptosis, Avian paramyxovirus-1, Avian virology, Chickens, Immunohistochemistry, In situ hybridization, Newcastle disease, Nucleotide sequence analysis, Pathogenesis, Pigeon paramyxovirus-1, Veterinary Pathology
VIRULENCE AND PATHOGENESIS OF NEWCASTLE DISEASE VIRUS
ISOLATES FOR DOMESTIC CHICKENS
M.S., Universidade Federal de Santa Maria, Brazil, 1993
A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial
Fulfillment of the Requirement for the Degree
DOCTOR OF PHILOSOPHY
ISOLATES FOR DOMESTIC CHICKENS
Committee: K. Paige Carmichael Barry Harmon
Bruce Seal Pedro Villegas
Electronic Version Approved:
Gordhan L. Patel Dean of the Graduate School The University of Georgia May 2002
iv
DEDICATION
I dedicate this work to my family. Your every day prayers and your love made me
stronger than I thought I could ever be. Muito obrigado!!!!!
v
ACKNOWLEDGMENTS
First of all, I would like to thank God for his care, for his love, and for his
answers to all my prayers over the last four years.
I would like to thank my major professors, Drs. Corrie Brown and Jack King, for
their expert guidance and patience with me and with this work. Both of them went far
beyond their call of duty as major professors, giving me their friendship and care.
I would like to thank all my committee members, Drs. Paige Carmichael, Barry
Harmon, Bruce Seal, and Pedro Villegas for their expert input and for all the time spent
reviewing this work. I would like to thank all the faculty of the Department of Pathology
for their teaching, patience, time, and for making me want to be an excellent pathologist
as all of them are.
I would like to thank all graduate students who shared sometime with me during
this journey. My special thanks goes to my first three officemates, Drs. Anapatricia
García, Lucia García-Camacho, and Suzana Tkalcic, and to my last three officemates,
Drs. Yong-Baek Kim, Laura Perkins, and Nobuko Wakamatsu. Without their friendship I
probably would not have done the work I came here to do.
I would like to thank all the staff members of the Department of Pathology. My
special thanks goes to Amanda Crawford, “my guardian angel”, who always stood by me
as a friend and cared for me as a member of her wonderful family. I would also like to
thank Erica Behling-Kelly, Joyce Bennett, Phillip Curry, Melissa Scott, and Dr. James
Stanton for their excellent technical assistance and patience in the laboratory.
vi
I would like to thank the Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq; Brasília, DF - Brazil), for supporting me with a scholarship. I wish
to thank the U.S. Poultry and Egg Association and USDA-ARS-CRIS for their support. I
also wish to thank Dr. David Swayne for allowing me to do a major part of my work in
the Southeast Poultry Research Laboratory (SEPRL - USDA).
I would like to thank all faculty of the Department of Pathology at the
Universidade Federal of Santa Maria (UFSM; Santa Maria – RS, Brazil). My special
thanks goes to Dr. Claudio Barros for all his efforts in helping me to be accepted as an
UGA PhD student.
My special thanks also goes to all my friends who were giving me their support,
sharing with me their experiences, and making me look forward to achieving my goals
along the last four years. This is a very long list represented here by the ones I have met
in the USA: Maria Rosa Ferreira, Eleonora and Marcos Machado, Monica and Carlos
Costa, Eugenia and Cármelo de Los Santos, and Carolina Realini.
vii
ISOLATES FOR DOMESTIC CHICKENS................................................... 52
5 VIRULENCE OF SIX HETEROGENEOUS-ORIGIN NEWCASTLE
DISEASE VIRUS ISOLATES BEFORE AND AFTER SEQUENTIAL
PASSAGES IN DOMESTIC CHICKENS ................................................... 124
6 PATHOGENESIS OF CHICKEN-PASSAGED NEWCASTLE
DISEASE VIRUSES ISOLATED FROM CHICKENS, WILD, AND
EXOTIC BIRDS ............................................................................................ 167
INTRODUCTION
Newcastle disease (ND) is one of the most important avian diseases because of its
potential economic impact on the poultry industry. Virulent ND is a reportable disease in
most countries of the world and is reportable as a List A disease for member countries of
the Office International des Epizooties (OIE).8 List A diseases are those that are highly
transmissible and their occurrence is of major importance for the international trade of
animals and animal products. 8 Newcastle disease is caused by an avian paramyxovirus
serotype 1 (APMV-1), synonymous with Newcastle disease virus (NDV).1, 2 NDV has
been classified according to five pathotypes that relate to the disease signs produced in
infected chickens: a) asymptomatic enteric NDV (avirulent viruses); b) lentogenic NDV
(low virulence viruses); c) mesogenic NDV (moderately virulent viruses); d) neurotropic
velogenic NDV (NVNDV; highly virulent neurotropic viruses); and e) viscerotropic
velogenic NDV (VVNDV; highly virulent viscerotropic viruses).1, 2, 6
There is an international trend that started in 1992 with the ND definition in the
European Union (EU) Council Directive 92/66/EEC,4 valid for countries belonging to the
EU, to separate NDV isolates into two virulence groups: the low virulence viruses
(asymptomatic enteric and lentogens) and the virulent viruses (mesogens and velogens),
based on the intracerebral pathogenicity index (ICPI) as a differential test.5
Following this international trend, the OIE established new standards during 1999
with a major change in the official definition of ND.7 ND is now defined as an infection
2
of birds caused by a virulent virus of the APMV-1 serotype. An isolate will be classified
as virulent for chickens if it has an ICPI of 0.7 or greater or if it has multiple basic amino
acids at the fusion (F) protein cleavage site.7
Currently, the United States Code of Federal Regulations (US-CFR) refers to the
presence of “exotic Newcastle disease” (END) as any velogenic ND. END is defined as
“an acute, rapidly spreading, and usually fatal viral disease of birds and poultry.” 3 A
major difference in the OIE’s definition for ND is that it refers to an infection with
viruses of the APMV-1 serotype, while the US-CFR refers to a disease caused by highly
virulent NDVs (velogenic pathotype).
In the USA, isolates that meet the OIE criteria for virulence (but do not meet the US-
CFR criteria) have been recovered almost exclusively from non-poultry sources,
including racing pigeons, wild, and exotic birds. The potential threat represented by these
isolates for poultry has to be thoroughly investigated because previous observations
revealed that NDV isolates from non-poultry species may not show their potential
virulence for domestic chickens in conventional pathogenicity tests, until passaged
several times in chickens. 1, 2
The difference between the OIE and US-CFR definitions of ND raises several
questions. What is the threat to poultry represented by isolates that are classified as
virulent by OIE but are not by the US-CFR? Could passage of these isolates in chickens
result in increased virulence that justifies using a more severe standard than the US-CFR?
What is the risk of trade sanctions if the USA has to adopt the OIE standards?
The overall hypothesis of this study was that the virulence of pigeon-origin and
heterogeneous-origin NDV isolates would increase after passage in domestic chickens.
3
To test this hypothesis, biological and molecular assays were performed to determine the
potential of six pigeon-origin and six primarily non-chicken-origin isolates of NDV to
cause disease in domestic chickens. These objectives were fulfilled by:
a) assessing the virulence of the NDV isolates before and after four serial passages in
chickens through four pathotyping tests: the intracerebral pathogenicity index (ICPI);
the intravenous pathogenicity index (IVPI); the mean death time (MDT); and the
intracloacal pathogenicity test.
b) evaluating portions of the viral genome, including the fusion (F) and matrix (M)
genes, before and after passage.
c) assessing the pathogenesis of the passaged viruses for chickens inoculated
intraconjunctivaly (a more natural route of infection) through evaluation of clinical
signs, mortality, gross and microscopic lesions, including immunohistochemistry, in
situ hybridization, and apoptosis assays.
REFERENCES
1. Alexander DJ: Newcastle disease and other avian Paramyxoviridae infections. In:
Diseases of Poultry, ed. Calnek BW, Barnes HJ, McDougall LR, and Saif YM, Beard
CW, 10th ed., pp. 541-569. Iowa State University Press, Ames, IA, 1997
2. Alexander DJ: Newcastle disease virus and other avian paramyxoviruses. In: A
laboratory manual for the isolation and identification of avian pathogens, ed. Swayne
DE, Glisson JR, Jackwood MW, Pearson JE, and Reed WM, 4th ed., pp. 156-163.
American Association of Avian Pathologists, Kennett Square, PA, 1998
4
3. Code of Federal Regulations (CFR), Title 9, Parts 1-199, Section 53.1. Cod Fed
Regulat 1:159, 2000
4. Council of the European Communities. Council Directive 92/66/EEC - introducing
Community measures for the control of Newcastle disease. Off J Europ Commun
35:1-20, 1992
5. King DJ: Newcastle disease virus pathotyping: the current emphasis on the ICPI.
Respiratory Diseases of Poultry - AAAP Symposium. Ann Conv Am Vet Med Assoc,
138:38-42, 2001
6. Office International des Epizooties: Manual of standards for diagnostic tests and
vaccines. Chapter 2.1.15 – Newcastle disease. 4th ed., pp. 221-232. OIE, Paris,
France, 2000
7. Office International des Epizooties: Official Acts. Resolution No. XIII - Newcastle
Disease. Off Int Epizoot Bull 111:266-267, 1999
8. Office International des Epizooties: OIE - Disease Classification. List A. Newcastle
disease. URL: www.oie.int/eng/maladies/en_classification.htm, January 10, 2002
CHAPTER 2
LITERATURE REVIEW
NEWCASTLE DISEASE
A. History
The first recorded outbreaks of Newcastle disease (ND) date from 1926 in Newcastle-
upon-Tyne, England 43 and on the island of Java, Indonesia.10 However, Alexander 12
mentioned the existence of evidence in the literature that there may have been outbreaks
of a ND-like disease before 1926. Possible problems in the assessment of ND outbreaks
before 1926 are the lack of distinctive clinical signs of ND and the difficulty in
differentiating ND from highly pathogenic avian influenza (HPAI) in the field because
both diseases cause similar respiratory signs and high mortality in avian species.12
B. Etiology
B. 1. Classification
Newcastle disease virus (NDV) is synonymous with avian paramyxovirus type 1
(APMV-1).10,11 It has been classified in the order Mononegavirales, family
Paramyxoviridae, subfamily Paramyxovirinae, genus Rubulavirus.10,11,89 However, the
complete nucleotide sequence of NDV showed that NDV is only distantly related to other
rubulaviruses. It has been proposed that NDV be considered as a member of a new genus
within the subfamily Paramyxovirinae.92 Based on matrix (M) protein amino acid
6
sequences, Seal et al 126 observed that NDV separates as a clade more closely related to
morbilliviruses than to rubulaviruses. A large-scale study of the phylogenetic
relationships among the Paramyxoviridae showed both fusion (F) and M phylogenies
were consistent with previous proposals for expanding the taxonomic diversity within the
Paramyxoviridae to include a new genus for NDV.140
NDV is a nonsegmented, single-stranded, negative-sense, enveloped RNA virus.
Viral particles are observed by electron microscopy as pleomorphic, varying from
spherical (150-300 nm in diameter) to filamentous (about 100 nm across and of variable
length). Projections of approximately 8-12 nm are observed on the viral surface,
corresponding to the F and hemagglutinin-neuraminidase (HN) glycoprotein spikes. The
“herring bone” nucleocapsid (about 13-18 nm in diameter) might be seen either free or
emerging from disrupted viral particles.10,89
The NDV genome consists of 15,156 nucleotides.98,115 Six major genes encode
the structural proteins (in the order 3’-NP-P-M-F-HN-L-5’): NP – nucleoprotein (an
RNA binding nucleocapsid protein); P – phosphoprotein (nucleocapsid associated); M -
matrix protein (an unglycosylated envelope protein); F - fusion protein (smaller surface
projections); HN – hemagglutinin-neuraminidase (larger projection on virus surface); and
L (large) protein – an RNA-dependent RNA polymerase associated with the
nucleocapsid.189,98,144
B. 2. Clinical forms
Specific clinical signs are not observed in birds with ND. The clinical disease might
range from subclinical infection to 100 % mortality in a short period of time. Many
7
factors related to the host (species, age, and immune status), virus strain (pathotype,
dosage and route of infection), and environmental or social stress can influence the
severity and the course of the disease.10,11,26,68
Historically, NDV has been classified according to five pathotypes that relate to the
disease signs produced in infected fully susceptible chickens:10,11,104 a) asymptomatic
enteric NDV - presence of avirulent viruses in the intestinal tract; b) lentogenic NDV -
mild or inapparent respiratory infection with low virulence viruses; c) mesogenic NDV -
low mortality, acute respiratory disease, and nervous signs in some birds infected with
moderately virulent viruses; d) neurotropic velogenic NDV (NVNDV) - respiratory and
neurologic signs with high mortality caused by highly virulent neurotropic viruses; and e)
viscerotropic velogenic NDV (VVNDV) - acute lethal infection caused by highly virulent
viscerotropic viruses in which hemorrhagic lesions are usually observed in the
gastrointestinal (GI) tract.10,11
According to the new ND definition105 (discussed at the end of this chapter), viruses
of all five pathotypes would fall into two major groups: “low virulence viruses” (for
asymptomatic enteric and lentogenic NDVs) and “virulent viruses” (for mesogenic and
velogenic NDVs).
B. 3. Biological and molecular characterization of NDV
Important concerns about NDV are related to the variation in virulence and in the
capacity to produce disease and how to differentiate between different types of NDV. 9,12
The conventional diagnosis of ND involves the isolation of the virus, the identification of
the virus as NDV, and the pathotyping tests to define the virulence of the isolate.10,11,104
8
Molecular-based techniques have also been used to assess the potential pathogenicity104
and for molecular epidemiology to determine the origin of the isolates and methods of
spread during ND outbreaks.2
Traditionally, in vivo tests have been used to make an acceptable assessment of the
virulence of NDV isolates for chickens.12 Four pathotyping tests have been employed to
differentiate virulence among NDV isolates: a) the intracerebral pathogenicity index
(ICPI) in 1-day-old chicks from specific-pathogen-free (SPF) parents; b) the intravenous
pathogenicity index (IVPI) in 6-week-old SPF chickens;10,11 and c) the intracloacal
inoculation pathogenicity test in 6-to-8-week-old chickens.10,11,113 The mean death time
(MDT) in 9-to-10-day-old embryonating eggs is another pathotyping test used during the
preliminary characterization of NDV virulence. Viruses are characterized as low,
moderate, or high virulence based on time to embryo death postinoculation, a more rapid
death indicating a more virulent virus.10,11 The ICPI differentiates low virulence lentogens
from mesogens of intermediate virulence and highly virulent velogens. In the ICPI test,
generally the lentogenic viruses give indices of up to 0.4, asymptomatic enteric viruses
usually slightly lower indices, mesogenic vaccines are usually around 1.4 and velogenic
viruses 1.7 upwards (maximum of 2.0).9 The IVPI differentiates most mesogens and all
lentogens from velogens 9,11 but tends not to show distinction between mesogenic and
lentogenic viruses.9 Isolates that produce severe disease and mortality following
intracloacal inoculation are identified as velogenic and VVNDV isolates are
differentiated from NVNDV by the presence of hemorrhagic lesions evident at
necropsy.11,113
9
Although the pathotyping tests have been employed successfully for NDV
characterization, they are laborious and there are some limitations and difficulties in
interpretation of the results.11 For example, standard ICPI and IVPI tests with PPMV-1
isolates produced compact grouping of the ICPIs but a wide range of IVPIs,17 making the
interpretation of the virulence of the viruses for chickens difficult. There is evidence that
viruses isolated from birds other than poultry may not show their “true virulence for
chickens” in conventional pathogenicity tests10,11 until passaged several times in
chickens.11 King 72 reported that the chicken breed in which the tests are performed
might also be a source of variability for the results of the pathotyping test. In that study it
was demonstrated that SPF White Leghorns were more susceptible than SPF White
Rocks. The accuracy of NDV virulence characterization is extremely important9 because
ND is a reportable disease that might result in international trade restrictions.104,108
The application of panels of monoclonal antibodies (MAbs) 6,15,90,112 was a major
advance for the characterization of NDV isolates, although it did not replace the in vivo
tests.2 Using conventional serologic tests with polyclonal antibodies, NDV isolates are
antigenically similar to the extent that all isolates are included in a single serotype. 11
However, MAbs may detect slight variations in antigenicity and provide an approach to
antigenic differentiation and grouping of NDV strains and isolates.2,16 The remarkable
uniqueness of the variant pigeon paramyxovirus-1 (PPMV-1) described below was
established through panels of Mabs.6,15,16,90,112
A severe disease in pigeons characterized clinically by diarrhea followed by nervous
signs that included ataxia, tremors, wing and leg paralysis, and torticollis spread through
most of Europe in the early 1980’s.134 The disease was caused by the NDV variant
10
PPMV-1, distinguishable from other members of the APMV-1 group by unique binding
patterns to monoclonal antibodies (MAbs).6,20,90,120 The sequence of events related to
PPMV-1 diagnosis and worldwide spread in pigeons is summarized on Table 2.1.
The pigeon-NDV variant was initially reported to be more virulent for pigeons than
for chickens.67 Further studies showed that despite the close antigenic identity among
them, PPMV-1 isolates have considerable variation in their pathogenicity for chickens.39
Turkeys experimentally infected with PPMV-1 presented similar clinical signs as
observed in chickens.66 PPMV-1 infection also has been reported in other birds species
such as kestrels (Falco tinnunculus)93 and pheasants (Phasianus colchicus).34
PPMV-1 was recognized as a very important disease-causing agent for domestic
poultry during 1984, when approximately 20 outbreaks of ND in non-vaccinated laying
hens in Great Britain were found to be caused by the NDV variant PPMV-1. The source
of the virus was probably feedstuffs contaminated with droppings and other materials
from infected feral pigeons feeding in certain feed stores in Liverpool docks.19,21 After
contact with infected pigeons, ND symptoms were also observed in poultry in Austria
during the 1980’s. 133
The importance of molecular-based techniques to characterize NDV isolates was
recognized by OIE during 1999,105…