Clinic for Poultry and · Bibliografische Informationen der Deutschen Bibliothek Die Deutsche...
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Bibliografische Informationen der Deutschen Bibliothek
Die Deutsche Bibliothek verzeichnet diese Publikation in der Deutschen Nationalbibliografie; Detaillierte bibliografische Daten sind im Internet über http://dnb.ddb.de abrufbar.
1. Auflage 2009
© 2009 by Verlag: Deutsche Veterinärmedizinische Gesellschaft Service GmbH, Gießen Printed in Germany
ISBN 978-3-939902-98-0
Verlag: DVG Service GmbH Friedrichstraße 17
35392 Gießen 0641/24466
[email protected] www.dvg.net
Clinic for Poultry and Anatomical Institute
University of Veterinary Medicine Hannover
Lectin and immuno histochemical investigations on cellular alterations in chicken embryos following inoculation with Newcastle Disease Virus (NDV) of
different virulence
THESISSubmitted in partial fulfillment of the requirements for the degree
DOCTOR OF PHILOSOPHY (PhD)
at the University of Veterinary Medicine Hannover
by
Julia Victoria Rodríguez Barahona
Costa Rica
Hannover, 2008
Supervisor: Prof. Dr. Neumann
Prof. Dr. Meyer
Advisory Committee: Prof. Dr. Liebler-Tenorio
Prof. Dr. Schumacher
PD. Dr. Grund
1st Evaluation: Prof. Dr. Neumann
Prof. Dr.Meyer
Prof. Dr. Liebler-Tenorio
Prof. Dr. Schumacher
2nd Evaluation: PD. Dr. Grund
Date of oral examination: 19 January 2009
Dios y la Virgen de los Angeles (Costa Rica)
”Printed with help from German Academic Exchange Service”
Contents Page
Index of Tables Index of Figures Abbreviations
I. Introduction 15
II. Literature review 171 Carbohydrates: General considerations 17
1.1 Structure and functions 17
1.2 Lectin definition and structure 20
1.3 Sugar specificity of lectins 20
1.4 Functions of lectins 22
1.5 Lectin histochemistry (LH) definition 23
1.6 LH for microscopy 24
1.6.1 Labels for detection 24
1.6.2 Methods for visualization 25
1.7 Studies in animals tissues 28
1.8 Used of lectins in virology studies (Glycovirology) 33
2 Immunohistochemistry (IHC) 352.1 General considerations 35
2.2 Studies with NDV 35
3 Newcastle Disease Virus (NDV) 393.1. General considerations 39
3.1.1 Virus characteristics 39
3.1.2 Epidemiology and geographic distribution 41
3.1.3 Prevention and control 41
3.1.4 Diagnostic 42
3.1.5 Incubation period, transmission routes and host range 43
3.1.6 Clinical signs 44
3.1.6 Histopathologic changes observed in chicken organs
infected with NDV.
45
3.2 Pathogenesis 46
3.2.1 Recognition and fusion mechanism 46
3.2.2 Virulence 48
4 Aims 53
III. Material and Methods 541 Introductory remarks 542 Determination of the LD50 of the NDV 55
2.1 Virus titration in cell culture (chicken fibroblasts) 55
2.2 Inoculation of embryonated chicken eggs with NDV 57
2.2.1 First viral passage 57
2.2.2 Second viral passage 58
3 Cytological studies 594 Studies of chicken embryos 60
4.1 Inoculation of chicken embryos 61
4.2 Embedding 62
4.2.1 Paraffin wax embedding 62
4.2.2 Technovit 7100 embedding 64
5 Histological staining 655.1 Paraffin sections 65
5.1.1 Hematoxylin and Eosin 65
5.1.2 Alcian Blue-PAS 65
5.1.3 Pappenheim´s staining (panoptic staining) 66
5.1.4 Immunohistochemistry 67
5.1.5 Lectin histochemistry 68
5.2 Technovit sections 71
5.2.1 Hematoxylin and Eosin 71
5.2.2 Toluidin blue 71
6 Data collection and statistical analysis 72
IV. Results 741 Determination of the LD50 of the NDV 74
1.1 Virus titration in cell culture (chicken fibroblasts) 74
1.2 Inoculation of embryonated chicken eggs 78
2 Cytological study 813 Studies of chicken embryos 82
3.1 Macroscopic observations 82
3.2 Results of the HA-Test 82
3.3 Immuno histochemical results 83
3.4 Histological and histochemical observations 85
3.4.1 Respiratory system 85
3.4.1.1 Histological observations 85
3.4.1.2 Lectin histochemical observations 87
3.4.2 Digestive system 92
3.4.2.1 Histological observations 92
3.4.2.2 Lectin histochemical observations 95
3.4.3 Immune system 104
3.4.3.1 Histological observations 104
3.4.3.2 Lectin histochemical observations 104
3.4.4 Urinary system (Kidney) 105
3.4.4.1 Histological observations 105
3.4.4.2 Lectin histochemical observations 105
3.4.5 Controls 106
V. Discussion 1091 Determination of the LD50 of the NDV 1092 Studies of chicken tissues 110
2.1 Macroscopical observations 110
2.2 Histological observations 111
2.2.1 Respiratory system 111
2.2.2 Digestive system 114
2.2.3 Immune system 121
2.2.4 Urinary system 123
2.3 Histopathological observations 124
2.4 Histochemical observations 125
2.4.1 Immuno histochemical observations 125
2.4.2 Lectin histochemical observations 129
3 Conclusions 138
VI. Summary (English) 141
VII. Summary (German) 143
VIII. Summary (Spanish) 145
IX. Literature cited 147
X. Annexes 170
Index of Tables
PageTable 1. Lectin histochemical reactions in organs of different animal species 29Table 2. Glycovirology studies realized for different viruses 34Table 3. Immuno histochemical studies for demonstration of NDV 37Table 4. Pathotypes and pathogenicity indices of NDV 41Table 5. Subgroups of embryonated chicken eggs inoculated 61Table 6. Lectins used in the study 69Table 7. Cytopathic effect observed in chicken fibroblast cultures infected
with NDV Herts33 75
Table 8. Cytopathic effect observed in chicken fibroblast cultures infected with NDV Komarov
76
Table 9. Cytopathic effect observed in chicken fibroblast cultures infected with NDV HB-1
77
Table 10. Mortality of chicken embryos in eggs inoculated with NDV Herts 33 78Table 11. Mortality of chicken embryos in eggs inoculated with NDV Komarov 79Table 12. Mortality of chicken embryos in eggs inoculated with NDV HB-1 79Table 13. Number of positive tissues infected with the different pathotype of
NDV by IHC. 83
Table 14. Lectin histochemical results of the respiratory system of uninfected chicken embryos of different days of incubation.
90
Table 15. Lectin histochemical results of the respiratory system of infected chicken embryos of different days of incubation.
91
Table 16. Lectin histochemical results of the digestive system of uninfected chicken embryos of different days of incubation.
99
Table 17. Lectin histochemical results of the digestive system of infected chicken embryos of different days of incubation.
102
Table 18. Lectin histochemical results of the immune system of uninfected chicken embryos of different days of incubation.
107
Table 19. Lectin histochemical results of kidney of uninfected chicken embryos of different days of incubation.
108
Index of Figures
PageFigure 1. Two major groups of glycoprotein sugar chains 18Figure 2. Visualizations methods in lectin histochemistry 27Figure 3. Viral proteins of NDV 40Figure 4. Life cycle of Influenza virus 48Figure 5. Localization of sections across the chicken embryo 63Figure 6. Survival time of chicken embryos infected with NDV at day 11th
of incubation 80
Figure 7. Results of HA-test of chicken embryos infected with NDV at day 11th of incubation
80
Figure 8. Immunocytochemistry of chicken fibroblasts 81Figure 9. Hemorrhagic lesions in chicken embryos infected with NDV 82Figure 10. Results of IHC in chicken infected embryos 84Figure 11. Trachea development of uninfected SPF chicken embryos 86Figure 12. Development of lung parabronchial tissues in uninfected SPF
chicken embryos 87
Figure 13. Esophagus development of uninfected SPF chicken embryos 93Figure 14. Ventriculus development of uninfected SPF chicken embryos 94Figure 15. Large intestine development of uninfected SPF chicken embryos 95Figure 16. Large intestine reaction of goblet cells in uninfected SPF chicken
Embryos 98
Figure 17. Bursa of Fabricius development from uninfected SPF chicken Embryos
105
Figure 18. Mucous gland development in the esophagus of the chick embryo
116
Figure 19. Diagram of successive stages in the structural and cytological development of the embryo chick
117
Figure 20. Stages in the development of glands of the ventriculus lining in the chick embryo
119
Figure 21. Glycosylation pattern of the cells of organs from uninfected chicken embryos
135
Figure 22. Glycosylation pattern of the cells of organs from uninfected and infected chicken embryos with Komarov pathotype at 15th i.d.
137
Abbreviations
ABC Avidin-biotin complex
AB-PAS Alcian blue-Periodic acid Schiff
AEC Amino ethyl carbozole
APAAP Alkaline phosphatase anti-alkaline phosphatase
AP Alkaline phosphatase
Asn Asparagine
BME Basal medium, Eagle
BSA-1 Bandeiraea simplicifolia
CAM Chorioallantoic membrane
Con A Concanavalin A
CPE Cytopathic effect
Cys Cystein
DAB Diaminobenzidine
DBP Dibutyl phthalate
ELISA Enzyme-linked immunosorbent assay
F Fusion protein
FBS Fetal bovine serum
FITC fluorescein isothiocyanate
fuc fucose
Gal galactose
galNac N-acetyl galactosamine
Glc glucose
glcNac N-acetyl glucosamine
Gln Glutamine
Gly Glycine
H 33 Herts 33
H&E Hematoxylin and Eosin
H2O2 Hydrogen peroxide
HA Hemagglutination
HB-1 Hitchner B1
HCl Acid chloride
HI Hemagglutination inhibition
HN Hemagglutinin-neuraminidase protein
i.d. Incubation day
IHC Immuno histochemistry
K Komarov
L Large protein
LD50 Lethal doses media
LH Lectin histochemistry
Lys Lysin
M Matrix protein
MAA Maackia amurensis agglutinin
Man mannose
N Nucleocapsid protein
NANA N-acetyl neuraminic acid
NDV Newcastle disease virus
NeuAc Neuraminic acid
OIE Office International of Epizooties
P Phosphor-protein
PAP Peroxidase anti-peroxidase
PBS Phosphate buffered saline
PCR Polymerase chain reaction
p.i. Post-inoculation
PO Peroxidase
RBCs Red blood cells
RPM Revolution per minute
RT-PCR Reverse transcriptase- PCR
Ser Serine
SPF Specific pathogen free
TB Toluidin blue
Thr Threonine
TRITC Tetramethylrhodamine isothiocyanate
WGA Triticum vulgaris agglutinin (Wheat Germ Agglutinin)
15
I. Introduction
Infection of cells with glycoproteins spikes containing viruses consist of various steps
including attachment of the virus to the host cell receptor, penetration, uncoating,
viral protein synthesis, glycosylation of viral proteins, transport by intracellular
trafficking, packaging, budding, and release of progeny viruses (SUZUKI, 2007).
Carbohydrates present on the cell surface are very diverse; their expression in
species, tissue, and individual cells is highly specific. Is important also consider, that
viruses have defined host range specificities. They recognize highly specific target
host cells and receptor molecules on the host cell surface. This suggests that viruses
may have been taking advantage of the diversity of host sugar chains to expand the
host range during evolution (SUZUKI, 2007).
By means of using the binding properties of lectins it is possible to define the
glycosylation pattern on cell surfaces. In this way lectin histochemistry is defined as
the binding of a lectin to tissue-bound carbohydrate residues, detected by means of a
visible label (BROOKS et al., 1997).
Regarding this approach, several investigations have already been conducted to
determine the normal lectin-binding properties in different tissues. For example, in
chicken the glycosylation patterns of air sacs (BEZUIDENHOUT, 2005), lung (GHERI
et al., 2000), choroids plexus (GHERI and SGAMBATI, 2003), lymphoid organs
(JÖRNS et al., 2003) and intestine (POHLMEYER, 2002) are described. Also, studies
in this regard have been conducted for tumor cells (BROOKS et al., 1997).
However, no information is available about glycosylation patterns of tissues following
infection with a virus with pathogenic potential for avian species. For this study, we
used Newcastle Disease Virus (NDV), of different pathotypes (lentogenic, mesogenic
and velogenic) as model.
16
This approach may give additional insights into the virus-cell interaction of NDV with
the host cell before and after its penetration and replication. This information may
also be of help to introduce antiviral therapy research employing lectins and their
corresponding sugars in future. In this context the distribution of free sugar moieties
in different organs of uninfected specific pathogens free (SPF) chicken embryos in
comparison to infected SPF chicken embryos was investigated.
NDV was selected because of its worldwide significance. The virus is classified in the
genus Avulavirus, sub-family Paramymovirinae, family Paramyxoviridae, order
Mononegavirales (ALEXANDER and JONES, 2008; KNIPE et al., 2007). The disease
affects principally poultry, is included in the List A of the Office International de
Epizooties (OIE) and requires obligatory reporting to this Office (OIE, 2004).
In addition, routine staining was employed like hematoxylin and eosin (H&E) staining
for histopathology, toluidine blue (TB) to recognize the presence of mucins, and
alcian blue-PAS (AB-PAS) for glycoconjugates. Immuno histochemistry (IHC) was
applied to confirm the presence of the virus in the tissues of NDV infected embryos
and, finally, lectin histochemistry (LH) to characterize the glycosylation pattern of the
tissues under investigation.
17
II. Literature review
1 Carbohydrates: General considerations
1.1 Structure and functions
The sugar chains in human and mammalian tissues are generally made up of
combinations of 7 simple sugars called monosaccharides. These are mannose
(man), glucose (glc), galactose (gal), fucose (fuc), N-acetyl galactosamine (galNac),
N-acetyl glucosamine (glcNac), and sialic or neuraminic acid (NeuAc or NANA)
(BROOKS et al., 1997).
When monosaccharides are covalently linked by a dehydration synthesis reaction
and specific enzymes, they form disaccharides or even longer sugar chains.
Oligosaccharides consist of a few covalently linked monosaccharides units, and are
often associated with proteins (glycoproteins) or lipids (glycolipids). Polysaccharides
comprise repeating monosaccharide or disaccharide units; usually they have
structural storage functions (BROOKS et al., 1997), for example glycogen in liver
(FRAPPIER, 2006).
Attachment of oligosaccharides to peptides increases solubility covers the antigenic
domains and protects the peptide backbones against proteases. Like polysialic acid
attached to neural cell adhesion molecules (N-CAM), the carbohydrate side chains
often modulate protein functions. In contrast, the carbohydrate moieties of serum
glycoproteins and pituitary glycoprotein hormones are involved in the clearance from
circulation or targeting of the hormones to respective organs (FURUKAWA, 1998).
18
Glycoproteins are macromolecules in which carbohydrates are attached covalently to
asparagine (N-glycans) or serine/threonine (O-glycans) residues of peptides (Fig. 1).
The N- and O-glycans are found in various cellular compartments and tissues
(FURUKAWA, 1998).
O-linked glycoproteins are often called mucin-type glycoproteins because this type of
linkage is typical for mucins. Mucins, which are very high molecular weight
glycoproteins with a high percentage of carbohydrate residues O-linked, are mostly
secreted by epithelial cells; however, they can also contain N-linked carbohydrate
residues (BROOKS et al., 1997).
In cells, glycoproteins and glycolipids are membrane associated or may sometimes
be cytoplasmic, and play an important functional role in cell-to cell interactions
(BROOKS et al., 1997; JÖRNS et al., 2003).
Figure 1. Two major groups of glycoprotein sugar chains (FURUKAWA, 1998, modified).
19
One important role of carbohydrates is that they can be recognized by proteins. For
example, "selectin" is expressed on endothelial cells or lymphocytes when leukocytes
migrate into the sites of inflammation or when mature lymphocytes migrate from or to
the blood circulation and to or from the lymphatic circulation (FURUKAWA, 1998).
They are also important in embryonal development. For example, during the
development of the mouse embryo, a dramatic change in the expression pattern of
cell surface antigens, which are mainly carbohydrates, is observed. Treatment of
embryos with haptenic sugars or biosynthetic inhibitors of glycosyltransferases or
processing glycosidases produce the arrest of development at certain stages,
suggesting that the carbohydrate antigens are essential for embryogenesis probably
by the involvement of specific cellular interactions (FURUKAWA, 1998).
As the carbohydrate, residues of the cell membrane are located at the extra cellular
surface of the membrane; one obvious specific function of the carbohydrate residues
is their interaction with naturally occurring ligands (BROOKS et al., 1997). For
example, sialoglycoproteins have three important functions; first, they act as
mediators of adherence by a negative charge. Second, they serve as cell surface
receptors for bacteria and viruses, and, third they have influence on viscoelastic
properties (YOON et al., 1998).
Alterations in the expression of sugar chains in several diseases can be recognized,
such as rheumatoid arthritis and cancer (BROOKS et al., 1997; FURUKAWA, 1998).
For these reasons, LH has been extensively used as an invaluable tool in mapping
cellular glycosylation.
20
1.2 Lectin definition and structure
The definition of the term ´lectin` adopted by the Nomenclature Committee of the
International Union of Biochemistry, and proposed by GOLDSTEIN et al. (1980) is: “a
carbohydrate binding protein of non-immune origin, which agglutinates cells and/or
precipitates polysaccharides or glycoconjugates”, and does not have an enzymatic
function.
Lectins selectively and specifically bind non-covalently to carbohydrate residues. An
important property of a lectin is the ability to bind to carbohydrates and, hence,
agglutinate cells, when no less than two binding sites are present (BROOKS et al.,
1997; HERNANDEZ et al., 1999; JÖRNS et al., 2003). It is for this reason that they
can be employed as specific probes to localize defined monosaccharides and
oligosaccharides on or in cells and the extracellular matrix.
Lectins have more than one binding site and therefore they are able to cross-link
cells through interactions with carbohydrates in the cell membrane. The amino acid
sequence of some lectins has been elucidated and its which are derived from human
and mammalian tissues appear to show a homology with plants lectins suggesting
that they have been highly conserved through evolution (BROOKS et al., 1997).
1.3 Sugar specificity of lectins
All lectins have a more or less well defined binding specificity; it is usually expressed
in terms of the simple monosaccharide which best inhibits its effect. Some examples
are shown as follows (DANGUY et al., 1998):
21
Lectin Inhibitory sugar Con A Glucose (Glc) and Mannose (Man)
WGA and BSA-II N-acetylglucosamin (GlcNac)
BSA-I and SBA N-acetylgalactosamin (GalNac) and galactose (Gal)
UEA-I L-fucose (L-fuc)
SNA and MAA Sialic acid (NANA)
Some carbohydrate structures are more common in nature than others. They are not
species specific (BROOKS et al., 1997), but organ-specific carbohydrates are
present. These findings provide the basis for evaluating why individual animals
acquired or required respective carbohydrate structures (FURUKAWA, 1998).
Some examples of different lectin families or subfamilies are shown below
(HIRABAYASHI, 2008):
(1) Ricin was the first lectin investigated in Russia more than 100 years ago. It is
now evident that ricin has many other homologous members which differ in
either toxicity or sugar-binding specificities.
(2) Galectins are a rapidly growing family of animal lectins; all of them share
galactose-specificity.
(3) Ca-dependent (C-type) animal lectins form an extremely large family,
composed of members having diverse structures and functions.
(4) Selectins form a distinguished subfamily of the C-type family by their specific
function in leukocyte adhesion to endothelial cells through sialyl-LewisX
recognition.
22
(5) Collectins, another subfamily of C-type lectins specific for mannose, have a
unique structure consisting of a C-type lectin domain and a collagen-like
domain. They are supposed to be involved in innate immunity.
(6) Invertebrates are known to contain various lectins in their body fluids,
probably as body-protection factors.
(7) Annexins are a group of proteins having affinity to lipids, but recently they
proved to be lectins showing a certain binding activity to glycosaminoglycans.
(8) The legume lectin family consists of a large number of members, such as Con
A, with variable saccharide specificity comparable to C-type lectins.
Galectins, C-type, annexins and invertebrate lectins are of animal origin and ricin and
the legume lectin family are plant lectins (HIRABAYASHI, 2008).
Cells and tissues contain and express a vast uncharted array of complex
carbohydrates in the form of glycoproteins, glycolipids, glycosaminoglycans, etc. The
role of these carbohydrates in cell communication and cell signaling events is
emerging and expanding the field of glycobiology (BROOKS et al., 1997).
1.4 Functions of lectins
Early workers proposed that plant lectins may represent a primitive defense system.
In this connection, many studies have demonstrated that plant lectins can recognize
and bind to complex carbohydrates on the surface of pathogenic microorganisms
(BROOKS et al., 1997).
23
Studies have also pointed out the involvement of animal lectins in cell adhesion and
recognition (BROOKS et al., 1997), as well as important proteins and glycoproteins
of the immune system (FUJITA et al., 2004). Regarding the last reference, some
authors investigated lectin reactions, for example, in marine sponges (Chondrilla
nucula) (MEYER et al., 2006), aquatic vertebrates (NONAKA 2001; SEKINE et al.,
2001; MEYER et al., 2007) and chicken (LYNCH et al., 2005).
Lectins of the legume family are reliable tools in genetic, biochemistry or immunologic
studies because, amongst others, they can bind different carbohydrates. They are
excellent for observing glycosylation of normal cells and tissues and changes
associated with cell behavior, development and disease (BROOKS et al., 1997,
HERNANDEZ et al., 1999).
Lectins can proof the presence of carbohydrates residues of glycoconjugates in the
cell membrane, in the extra cellular matrix and alterations of the cellular surface
(HERNANDEZ et al., 1999). In tissue affected by storage diseases or in cancer cells,
it is possible to observe an increase in lectin binding (BROOKS et al., 1997), for
example, investigation of adenocarcinomas in the human lung (THÖM et al., 2007).
It is also possible to investigate the interaction between virus and the cell receptor,
for example, Influenza virus (ZHANG et al., 2005) and Adenovirus (WU et al., 2006),
see chapter 1.8. Lectins can be used to observe glycosylation patterns during
physiologic phenomenons like pregnancy (PEEL et al., 1996), or organ development
(FERNANDEZ et al., 1994).
1.5 Lectin histochemistry (LH) definition
Lectin histochemistry is defined as the binding of a lectin to tissue-bound
carbohydrate residues, detected by means of a visualization system (for example
24
DAB-PO). Technically, three criteria have to be ful filled to employ LH successfully
(BROOKS et al., 1997):
(1) A suitable lectin has to be selected: Lectins are commercially available; they
have to be chosen, according to the specific sugar spectrum that has to be
studied, as related to the aims of the question to be investigated. Uses of
positive and negative controls are indispensable to standardize a LH
technique.
If one is looking for O-linked glycoproteins is also important to select lectins
with binding specificities to the following carbohydrate moieties: galNac,
glcNac, gal, fuc and NANA. For N-linked glycans, it is preferable to use
lectins, which recognize mannose (see chapter 1.2).
(2) For the preservation of carbohydrate structures in the cell or the tissues it is
important to consider that enzyme digestion can damage or strip
glycoproteins of interest from the cell surface, thus altering the results of
lectins binding studies. Generally speaking, carbohydrate structures are
relatively resistant and can be damaged by fixation or processing.
(3) Selection of a visible label, for example avidin-biotin labeling (discussed in
chapter 1.6).
1.6 LH for microscopy
1.6.1 Labels for detection
To choose a convenient label for LH it is important to consider tissue type, difficulties
of interpretation and endurance of the label. Labels existing for lectin detection are
divided into two principal categories: fluorescent labels and enzyme labels.
25
As in immunofluorescence, fluorescent labels fluoresce brightly when viewed under
exiting, employing the appropriate filter, ultraviolet or high-energy blue light.
Conventional fluorescent labels include FITC, which fluoresces green/yellow and
TRITC or Texas Red, which fluoresces red. The limitations of this labels are that the
fluorescence signal will remain strong for only a few hours, or in some cases a few
days, and it is sometimes difficult to identify the exact cell type or structure (because
unlabeled cells remain invisible) (BROOKS et al., 1997).
The most commonly enzyme labels for LH used are horseradish PO and AP. Other
enzyme labels are less commonly used. They are detected through their reaction
with a colorless chromogenic substrate to yield a color product. Examples of
chromogenic substrates using peroxidase are DAB and AEC, and for AP naphthol-
AS-BI-phosphate and Fast Red or Fast Blue. Limitations for these labels are that
many tissues contain appreciable amounts of endogenous peroxidase and/or alkaline
phosphatase, and they will show the same color reaction with the chromogenic
substrate. Advantages of this label are that the interpretation of results is easier and
the sections are enduring (BROOKS et al., 1997).
1.6.2 Methods for visualization
BROOKS et al. (1997) described the following methods for visualization: (1) direct,
(2) simple indirect, (3) APAAP or PAP and (4) Avidin-biotin.
(1) The direct method is the most simple and straightforward method. It makes
use of lectin, which is directly conjugated, to a fluorescent or enzyme label.
With this method, the cell/tissue preparation is incubated with the directly
labeled lectin, it binds to the glycoconjugates expressed by the cells and
binding is visualized by the label directly attached to it. This method is less
sensitive on tissue than the more complex multi-step techniques (see Fig.
2.A).
26
(2) Simple indirect methods are uncomplicated and rapid, which has the
advantage that unconjugated lectins are used. In this case, the sample is
incubated first with a lectin, which is detected by a labeled antibody directed
against the lectin. Polyclonal antiserum is commercially available, usually
raised in rabbits, either unlabeled or labeled with fluorescence tags,
horseradish peroxidase or alkaline phosphatase (Fig. 2.B).
(3) APAAP or PAP methods are similar, both are multi-step methods that give
good results and are very sensitive. The difference is that the first one uses
alkaline phosphatase as label and the second one horseradish peroxidase.
Employing these methods, the sample is incubated with the unlabeled lectin,
then with the unlabeled rabbit antibody against the lectin, in the next step a
linking antibody is added and subsequently an APAAP or PAP complex (Fig.
2.C).
(4) Avidin-biotin methods are commercially available and conjugated to
fluorescent labels, alkaline phosphatase and horseradish peroxidase. There
are three different techniques: (1) biotinylated lectin (Fig. 2.D), (2) biotinylated
antibody (Fig. 2.E) and (3) ABC (Fig. 2.F). The first one is simple, cheap and
offers a sensitive, fast and very straightforward method for detecting
carbohydrate residues. The second one uses an unlabeled lectin and a biotin-
labeled avidin or streptavidin. And the last one is a much more complex multi-
step technique that has the advantage of being extremely sensitive; here an
unlabelled lectin is layered with an unlabelled primary polyclonal rabbit
antibody against the lectin. Next, a biotinylated second polyclonal antibody
raised in swine against rabbit immunoglobulin is added. Finally, a labeled pre-
formed complex of avidin and biotin mix is added.
27
Figure 2. Visualizations methods in lectin histochemistryA= direct, B= simple indirect, C= APAAP or PAP, D= Biotinylated lectin, E= Biotinylated antibody, F= ABC. a= carbohydrate, b= lectin, c= label, d= antibody against the lectin, e= rabbit anti lectin, f= swine anti rabbit, g= APAAP or PAP complex, h= biotin, i= avidin.
28
1.7 Studies in animals tissues
Under normal conditions, glycosylation patterns may be changed according to the
animal species, organ, developmental stage of the animal, physiological phenomena,
etc. For this motive, some studies were conducted to determinate the glycosylation
pattern in different animals and organs. Examples see Table 1.
29
Tabl
e 1.
Lect
in h
isto
chem
ical
reac
tions
in o
rgan
s of
diff
eren
t ani
mal
spe
cies
Le
ctin
Org
an o
r tis
sue
Spec
ieR
efer
ence
Thym
us,
eryt
hroc
ytes
, bu
rsa
of
Fabr
iciu
s,
sple
en, b
one
mar
row
, lun
g, li
ngua
l gla
nds
and
chor
ioal
lant
oic
mem
bran
e
Chi
cken
GH
ER
I et a
l., 1
992;
FE
RN
AN
DE
Z et
al.,
199
4;
GH
ER
I et a
l., 2
000;
GA
BR
IELL
I et a
l, 20
03; J
ÖR
NS
et
al.,
200
3; M
OR
AE
S a
nd M
ELL
O, 2
006
Inte
stin
eC
hick
enan
d m
ouse
KA
ND
OR
I et a
l., 1
996;
KIT
AG
AW
A e
t al.,
200
0;
PO
HLM
EY
ER
, 200
2
Hep
atoc
ytes
Mou
seM
OR
AE
S a
nd M
ELL
O, 2
006
Con
A
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
Thym
us,
inte
stin
e an
d ch
orio
alla
ntoi
c m
embr
ane
Chi
cken
FER
NA
ND
EZ
et a
l, 19
94; K
ITA
GA
WA
et a
l., 2
000;
G
AB
RIE
LLI e
t al,
2003
R
CA
-I
Em
bryo
Mou
seM
IOS
GE
et a
l, 19
97
Thym
us;
burs
a of
Fa
bric
ius,
sp
leen
, bo
ne
mar
row
, lu
ng,
inte
stin
e,
lingu
al
glan
ds
and
chor
ioal
lant
oic
mem
bran
e
Chi
cken
GH
ER
I et a
l., 1
992;
FE
RN
AN
DE
Z et
al,
1994
; K
AN
DO
RI e
t al.,
199
6; G
HE
RI e
t al.,
200
0;
KIT
AG
AW
A e
t al.,
200
0; P
OH
LME
YE
R, 2
002;
G
AB
RIE
LLI e
t al,
2003
JÖR
NS
et a
l., 2
003
Em
bryo
Mou
seM
IOS
GE
et a
l, 19
97
WG
A
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
Thym
us,
lung
,
lingu
al
glan
ds
and
chor
ioal
lant
oic
mem
bran
e C
hick
enG
HE
RI
et
al.,
1992
; FE
RN
AN
DE
Z et
al
, 19
94;
GH
ER
I et a
l., 2
000;
GA
BR
IELL
I et a
l, 20
03
LTA
Em
bryo
Mou
seM
IOS
GE
et a
l, 19
97
Thym
us,
burs
a of
Fa
bric
ius,
sp
leen
, bo
ne
mar
row
, lu
ng,
lingu
al
glan
ds
and
chor
ioal
lant
oic
mem
bran
e
Chi
cken
GH
ER
I et a
l., 1
992;
GH
ER
I et a
l., 2
000;
G
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3 U
EA-I
Inte
stin
eC
hick
en,m
ouse
and
rabb
it K
AN
DO
RI e
t al.,
199
6; G
EB
ER
T an
d P
OS
SE
LT,
1997
; KIT
AG
AW
A e
t al.,
200
0 A
UA
, Cals
epa,
NPA
, ML-
I, PT
A, C
odium
, HPA
, ML-
III,
STA,
Vis
cal
Thym
us,
burs
a of
Fab
riciu
s, s
plee
n an
d bo
ne
mar
row
Chi
cken
JÖR
NS
et a
l., 2
003
30
… c
ontin
uatio
n Ta
ble
1.Le
ctin
his
toch
emic
al re
actio
ns in
org
ans
of d
iffer
ent a
nim
al s
peci
esLe
ctin
Org
an o
r tis
sue
Spec
ieR
efer
ence
Cona
rva,
ML-
II, W
FA,
DSA
, LEA
, PSA
, AIA
Thym
us,
burs
a of
Fa
bric
ius,
sp
leen
, bo
ne
mar
row
and
inte
stin
e C
hick
enK
ITA
GA
WA
et a
l., 2
000;
PO
HLM
EY
ER
, 200
2 ;
JÖR
NS
et a
l., 2
003
Thym
us, b
ursa
of F
abric
ius
and
bone
mar
row
C
hick
enJÖ
RN
S e
t al.,
200
3 G
NA,
HH
A, L
OA,
AC
A,
APA
,BD
A, C
AA
, IR
AS
plee
nC
hick
en a
nd ra
t D
ÜLL
MA
N e
t al.,
200
0; J
ÖR
NS
et a
l., 2
003
Thym
us,
burs
a of
Fa
bric
ius,
S
plee
n,
bone
m
arro
wC
hick
enJÖ
RN
S e
t al.,
200
3 LC
A
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
Thym
us,
burs
a of
Fab
riciu
s, s
plee
n an
d bo
ne
mar
row
Chi
cken
JÖR
NS
et a
l., 2
003
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
BSA-
I
Inte
stin
eM
ouse
KA
ND
OR
I et a
l., 1
996
ECA
Thym
us,
burs
a of
Fa
bric
ius,
sp
leen
, bo
ne
mar
row
and
cho
rioal
lant
oic
mem
bran
e C
hick
enG
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3
Thym
us,
burs
a of
Fa
bric
ius,
sp
leen
, bo
ne
mar
row
and
inte
stin
e C
hick
enP
OH
LME
YE
R, 2
002;
JÖ
RN
S e
t al.,
200
3 M
PA, P
HA
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
Thym
us,
burs
a of
Fab
riciu
s, s
plee
n an
d bo
ne m
arro
w
Chi
cken
JÖR
NS
et a
l., 2
003
BPA
Inte
stin
eM
ouse
KA
ND
OR
I et a
l., 1
996
Thym
us, b
ursa
of F
abric
ius,
bon
e m
arro
w,
lung
and
ling
ual g
land
s C
hick
enG
HE
RI e
t al.,
199
2; G
HE
RI e
t al.,
200
0;
JÖR
NS
et a
l., 2
003
Spl
een
Chi
cken
and
rat
DÜ
LLM
AN
et a
l., 2
000;
JÖ
RN
S e
t al.,
200
3
SBA
Inte
stin
eC
hick
enan
dm
ouse
KA
ND
OR
I et a
l., 1
996;
KIT
AG
AW
A e
t al.,
200
0
31
… c
ontin
uatio
n Ta
ble
1. L
ectin
his
toch
emic
al re
actio
ns in
org
ans
of d
iffer
ent a
nim
al s
peci
es
Le
ctin
Org
an o
r tis
sue
Spec
ieR
efer
ence
Thym
us, i
ntes
tine
Chi
cken
and
m
ouse
KA
ND
OR
I et a
l., 1
996;
KIT
AG
AW
A e
t al.,
20
00; J
ÖR
NS
et a
l., 2
003;
ALV
AR
EZ
et a
l.,
2006
Bur
sa o
f Fab
riciu
s, s
plee
n, b
one
mar
row
, lu
ng, c
horio
alla
ntoi
c m
embr
ane
and
nasa
l ca
vity
Chi
cken
YO
ON
et a
l., 1
998;
GH
ER
I et a
l., 2
000;
G
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3
Ling
ual g
land
s
Chi
cken
,m
ouse
, rat
an
d ha
mst
er
SH
ULT
E a
nd S
PIC
ER
, 198
5; G
HE
RI e
t al.,
19
92
Trac
hea
and
panc
reas
M
ouse
, rat
an
d ha
mst
er
SH
ULT
E a
nd S
PIC
ER
, 198
5
PNA
Olfa
ctor
y ep
ithel
ium
R
atH
EM
PS
TEA
D a
nd M
OR
GA
N, 1
983
Thym
us, b
ursa
of F
abric
ius,
bon
e m
arro
w,
lung
, na
sal
cavi
ty
and
chor
ioal
lant
oic
mem
bran
e
Chi
cken
YO
ON
et a
l., 1
998;
GH
ER
I et a
l., 2
000;
G
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3
Spl
een
Chi
cken
and
rat
DÜ
LLM
AN
et a
l., 2
000;
JÖ
RN
S e
t al.,
200
3
Inte
stin
eC
hick
enan
dm
ouse
KA
ND
OR
I et a
l., 1
996;
KIT
AG
AW
A e
t al.,
200
0
Ling
ual g
land
s
Chi
cken
, rat
, m
ouse
and
ha
mst
er
SH
ULT
E a
nd S
PIC
ER
, 198
5; G
HE
RI e
t al.,
19
92
DBA
Trac
hea
and
panc
reas
R
at, m
ouse
an
d ha
mst
er
SH
ULT
E a
nd S
PIC
ER
, 198
5
Thym
us,
burs
a of
Fab
riciu
s, b
one
mar
row
an
d in
test
ine
Chi
cken
PO
HLM
EY
ER
, 200
2; J
ÖR
NS
et a
l., 2
003
CM
A, U
DA
Spl
een
Chi
cken
and
rat
DÜ
LLM
AN
et a
l., 2
000;
JÖ
RN
S e
t al.,
200
3
32
… c
ontin
uatio
n T
able
1.
Lect
in h
isto
chem
ical
reac
tions
in o
rgan
s of
diff
eren
t ani
mal
spe
cies
Le
ctin
Org
an o
r tis
sue
Spec
ieR
efer
ence
MA
ATh
ymus
, bu
rsa
of F
abric
ius,
spl
een,
bon
e m
arro
w,
na
sal
cavi
ty,
inte
stin
e an
d co
rioal
lant
oic
mem
bran
e
Chi
cken
YO
ON
et a
l., 1
998;
PO
HLM
EY
ER
, 200
2;G
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3
Thym
us, b
ursa
of F
abric
ius,
bon
e m
arro
w,
nasa
l ca
vity
, in
test
ine
and
chor
ioal
lant
oid
mem
bran
e
Chi
cken
YO
ON
et a
l., 1
998;
PO
HLM
EY
ER
, 200
2;G
AB
RIE
LLI e
t al,
2003
; JÖ
RN
S e
t al.,
200
3 SN
A
Spl
een
Chi
cken
and
rat
DÜ
LLM
AN
et a
l., 2
000;
JÖ
RN
S e
t al.,
200
3
ASA
Spl
een
Rat
DÜ
LLM
AN
et a
l., 2
000
ALL
Thym
usM
ouse
ALV
AR
EZ
et a
l., 2
006
SJA,
LC
A In
test
ine
Chi
cken
KIT
AG
AW
A e
t al.,
200
0 VV
AIn
test
ine
Chi
cken
and
rabb
itG
EB
ER
T an
d P
OS
SE
LT, 1
997,
KIT
AG
AW
A e
t al
., 20
00
Con
A=
Con
cana
valia
ens
iform
is,
RC
A-I=
Ric
inus
com
mun
is,
WG
A=
Triti
cum
vul
garis
, LT
A=
Tetra
gono
lobu
s pu
rpur
ea,
UEA
-I= U
lex
euro
paeu
s,A
UA
= Al
lium
urs
inum
,Ca
lsepa
=Ca
lyste
gia s
epium
agg
lutini
n, N
PA=
Narc
issus
pse
udon
arcis
sus,
ML
(I-III)
= Vi
scum
albu
m, P
TA=
Psop
hcar
pus
tetra
gono
lobus
, Cod
ium,
HPA
=H
elix
pom
atia
, ST
A=So
lanu
m t
uber
osum
, Vi
scal
= Vi
scum
alb
um, C
onar
va=
Conv
olvulu
s ar
vens
is ag
glitin
in,
WFA
= W
iste
ria f
lorib
unda
, D
SA=
Dat
ura
stra
mon
ium
, L
EA=
Lyco
pers
icon
esc
ulen
tum
, PS
A= P
isum
sat
ivum
, AI
A= A
rtuca
rpus
int
egrif
olia
, G
NA=
Gal
anth
us n
ival
is,
HH
A= H
elix
aspe
rsa,
LO
A= L
athy
rus
odor
atus
, AC
A= A
mar
anth
us c
auda
tus,
APA
= Ab
rus
prec
ator
ius,
BD
A=
Bry
onia
dio
ica,
CA
A=
Car
agan
a ar
bore
scen
s, IR
A=
Iris
retic
ulat
a,LC
A=
Lens
cul
inar
is,
BS (
I-II)=
Ban
deira
ea s
impl
icifo
lia,,
ECA=
Ery
thrin
a cr
ista
galli,
MPA
= M
aclu
ra p
omife
ra,
PHA=
Pha
seol
us v
ulga
ris, P
NA=
Ara
chis
hypo
gaea
, BPA
= Ba
uhin
ia p
urpu
rea,
DB
A=
Dol
icho
s bi
floru
s, S
BA=
Gly
cine
max
ima,
CM
A= C
helid
oniu
m m
ajus
, U
DA=
Urti
ca d
ioic
a, M
AA
= M
aack
ia
amur
ensi
s, S
NA
= S
ambu
cus
nigr
a, A
SA=
Alliu
m s
ativ
um, A
LL=
Am
aran
thus
leuc
ocar
pus,
SJA
= S
opho
ra ja
poni
ca, L
CA=
Len
s cu
linar
is, V
VA=
Vic
ia
villo
sa.
33
1.8 Use of lectins in virology studies (Glycovirology)
Viruses evade the immune defense systems of the host by adding sugar chains to
their spike proteins and in some cases gain easy entry into host cells by being
trapped by lectins that recognize host cell sugar chains. The viral envelope consists
of host-derived phospho- and glycolipids, complex lipids including cholesterol, and
virus-specific glycoprotein spikes. These spikes play essential roles in viral adhesion
to the host and viral release from the host after budding (SUZUKI 2007).
Attachment of the virus to the host receptor, penetration, uncoating, viral protein
synthesis, glycosylation, transport by intracellular trafficking, packaging, budding and
release of progeny viruses are steps required to the transmission process of viruses
with glycoprotein spikes into host cells (SUZUKI 2007).
Host cell sugar chains are very diverse but the expression of every sugar chain in
species, tissue, and individual cells is highly specific. On the other hand, viruses
have defined host range specificity and recognize target host cells and receptor
molecules highly specific on the host cell surface. This fact suggests that viruses may
have been taking advantage of the diversity of host sugar chains to expand the host
range during evolution, and the high specificity of viruses for recognition of the target
host cell receptor may reflect the highly specific expression of sugar chains by host
cells. It has been reported that modification of sugar chains on the viral spike protein
is not only closely related to viral infectivity but also to the pathogenesis of viral
diseases (SUZUKI, 2007).
Many enveloped viruses recognize and bind to sugar chains on the host cell
membrane, acting as specific viral receptors. Some studies have been conducted in
order to evaluate the receptor on tissues infected with influenza virus, Human
parainfluenza virus type 3, Adeno-Associated virus types 1 and 6, Herpes simplex
virus type I, Enterovirus 70, Feline calicivirus, Avian Infectious bronchitis virus and
Newcastle Disease Virus, some examples are mentioned in Table 2.
34
Table 2. Glycovirology studies realized for different virusesLectin Virus Reference
MAA Influenza virus
Human parainfluenza virus type 3
Adeno-Associated Virus Types 1 and 6
Herpes simplex virus type I
Enterovirus 70
Feline calicivirus
Avian Infectious bronchitis virus
WAN and PEREZ, 2005
ZHANG et al, 2005
WU et al, 2006
TEUTON and BRANDT, 2007
NOKHBEH, 2005
STUART and BROWN, 2007
WINTER et al, 2006
SNA Influenza virus
Human parainfluenza virus type 3
Adeno-Associated Virus Types 1 and 6
Enterovirus 70
Feline calicivirus
WAN and PEREZ, 2005
ZHANG et al, 2005
WU et al, 2006
NOKHBEH, 2005
STUART and BROWN, 2007
ELD Herpes simplex virus type I TEUTON and BRANDT, 2007
WGA Feline calicivirus STUART and BROWN, 2007
Feline calicivirus STUART and BROWN, 2007 Con A
Newcastle Disease Virus McMILLAN et al., 1985
SBA, PHA, DBA, LCA, PSA, LTA
Newcastle Disease Virus McMILLAN et al., 1985
PNA, HPA Influenza virus LUTHER et al., 1980
MAA=Maackia amurensis, SNA=Sambucus nigra, ELD=Elderberry bark, WGA=Triticumvulgaris, Con A=Concanavalia ensiformis, SBA=Glycine max, PHA=Phaseolus vulgaris,DBA=Dolichos biflorus, LCA=Lens culinaris, PSA=Pisum sativum, LTA=Tetragonolobuspurpureas, PNA=Arachis hypogaea, HPA=Helix pomatia.
35
2. Immunohistochemistry (IHC)
2.1. General considerations
Immunohistochemistry is the recognition of innate proteins or antigens in tissue
sections through the antigen-antibody reaction by the use of marker labeled
antibodies as specific reagents; this marker can be fluorescent dyes, enzymes,
radioactive elements or colloidal gold (http://www.ihcworld.com/_intro/intro.htm).
This technique is considered as a powerful method for localizing a specific antigen to
particular cell types in a heterogeneous population or to specific compartments within
a cell (LU et al., 1998).
The sensitivity of the technique is much enhanced by amplification systems involving
indirect detection of the primary antibody by one or more additional steps. However,
a limitation of indirect immuno histochemistry is that primary antibody raised in a
given species usually cannot be applied to tissues from the same species (LU et al.,
1998). Labels and methods of visualization are the same as described in Chapter 1.6
(LH for microscopy).
2.2. Studies with NDV
The selection of the primary antibody is one of the most important steps to obtain
good results by IHC techniques. The antibodies used for specific detection can be
polyclonal or monoclonal. Monoclonal antibodies are generally considered to exhibit
greater specificity. A polyclonal antibody is made by injecting animals with peptide
antigens, and then after a secondary immune response is stimulated, isolating
antibodies from whole serum. Thus, polyclonal antibodies are a heterogeneous mix
36
of antibodies that recognize several epitopes (http://www.answers.com/
immunohistochemistry).
Polyclonal and monoclonal antibodies against NDV are used for different techniques
like Hemagglutination Inhibition (HI) test, virus neutralization (MEULEMANS et al.,
1987; LANA et al., 1988; ALAMARES et al., 2005), radioimmunoprecipitation,
infected cells surface and cytoplasmic fluorescence (IORIO and BRATT, 1983) and
ELISA (ALAMARES et al., 2005). Examples of studies conducted to determine the
efficacy of different antibodies to detect the NDV with IHC techniques are mentioned
in Table 3. More information regarding virus characteristics are described in chapter
3.
37
Tabl
e 3.
Imm
uno
hist
oche
mic
al s
tudi
es fo
r dem
onst
ratio
n of
ND
V A
ntib
ody/
Ani
mal
In
ocul
atio
n vi
a/
viru
lenc
e/
viru
s st
rain
O
rgan
Ref
eren
ce
IgG
m
onoc
lona
l ag
ains
t ph
osph
opro
tein
/ S
PF
Whi
te L
egho
rns
Chi
cken
Intra
ocul
arly
, int
ratra
chea
lly/
M=R
oaki
nTr
ache
a (+
), lu
ng (+
), sp
leen
(-),
Har
deria
n gl
and
(-) a
nd
ceca
l ton
sil (
-).
LOC
KA
BY
et
al.,
199
3
Coc
ktai
l m
onoc
lona
l (1
5C4+
Q24
) /S
PF
Whi
te R
ock
chic
ken
Con
junt
ival
sa
c/
VV
=Cal
iforn
ia
1083
, In
done
sian
co
ckat
oo,
Chi
nese
par
akee
t
Eye
lid (
+),
sple
en (
+),
burs
a of
Fab
riciu
s (-)
, sm
all
inte
stin
e (+
), ce
cum
(+).
BR
OW
N
et
al.,
1999
Coc
ktai
l m
ouse
m
onoc
lona
l (7
9, 1
5C4
and
Q24
)/S
PF
Whi
te R
ock
chic
ken
Con
junt
ival
sac
/V
V=C
alifo
rnia
10
83,
90-1
4698
(In
done
sian
co
ckat
oo),
93-
2871
0 (C
hine
se
para
keet
), V
N=T
exas
G
B,
Turk
ey
ND
, M
=Roa
kin,
A
nhin
ga,
L=B
H-1
an
d Q
V4.
Eye
lid (
VV
+),
sple
en (
VV
+),
air
sac
(VV
+),
lung
(V
V+)
, he
art
(VV
+),
ceca
l ton
sil (
VV
+) a
nd b
rain
(V
V+)
. O
ther
pa
thot
ypes
was
ver
y w
eak
and/
or in
cons
iste
nt.
BR
OW
N
et
al.,
1999
Rab
bit
anti-
pep
tide
vira
l N
pr
otei
n/S
PF
Whi
te L
egho
rns
Chi
cken
Intra
mus
cula
rly
/ M
=Anh
inga
, P
heas
ant,
and
dove
, L=
C
hick
en
Live
B
ird
mar
ket,
Yel
low
N
ape
parr
ot
and
Ckn
-A
ustra
lia.
Com
b (M
+), s
plee
n (-
), th
ymus
(M
+), b
ursa
(M
+), e
yelid
(M
+,
L+),
Har
deria
n gl
and
(M+)
, or
opha
rynx
(L
+),
esop
hagu
s (-
), pr
oven
ticul
us (
-),
panc
reas
(M
+),
smal
l in
test
ine
(-),
ceca
l ton
sils
(-)
, la
rge
inte
stin
e (+
), ca
udal
th
orac
ic a
ir sa
c (-
), tra
chea
(M
+), l
ung
(M+)
, hea
rt (M
+),
liver
(M
+),
kidn
ey (
M+)
, br
east
and
thi
gh m
uscl
es (
-),
scia
tic n
erve
s (-
), br
ain
(M+)
and
bon
e m
arro
w (M
+).
KO
MM
ER
Set
al.,
200
3
Rab
bit
anti-
pep
tide
vira
l N
pr
otei
n /
SP
F W
hite
Leg
horn
s C
hick
en
Cho
rioal
lant
oic
mem
bran
e/
VV
=Fon
tana
, C
alifo
rnia
10
83;
VN
=Bea
udet
te;
M
=Roa
kin,
A
nhin
ga; L
=QV
4, L
aSot
a;
CA
M (L
+, M
+, V
V+,
VN
+), e
soph
agus
(L+)
, air
sac
(L+)
, lu
ng (
M+,
VV
+, V
N+)
, m
uscl
e (M
+, V
V+,
VN
+),
kidn
ey
(M+,
VV
+, V
N+)
, ski
n (M
+, V
V+,
VN
+).
OLD
ON
I et
al
., 20
05
38
…..
cont
inua
tion
Tabl
e 3.
Imm
uno
hist
oche
mic
al s
tudi
es fo
r dem
onst
ratio
n of
ND
VA
ntib
ody/
Ani
mal
In
ocul
atio
n vi
a/
viru
lenc
e/
viru
s st
rain
O
rgan
Ref
eren
ce
Rab
bit
anti-
pep
tide
vira
l N
pr
otei
n/
chic
kens
, S
PF
turk
eys,
com
mer
cial
tur
keys
, pi
geon
s
Intra
conj
untiv
ally
an
d in
trana
sally
/
Chi
cken
C
A/S
0212
676
Eye
lid (+
), sp
leen
(+),
thym
us (+
), bu
rsa
(+),
Har
deria
n gl
and
(+),
prov
entri
culu
s (+
), sm
all i
ntes
tine
(+),
Mec
kel’s
div
ertic
ulum
(+),
ceca
l ton
sils
(+),
larg
e in
test
ine
(+),
air s
ac (+
), tra
chea
(+),
lung
(+),
hear
t (+)
, es
opha
gus
(+),
tong
ue/p
hary
nx (+
), cr
op (+
), br
ain
(+),
liver
(+),
kidn
ey (+
), co
mb-
only
from
chi
cken
s (+
), bo
ne
mar
row
(+),
and
turb
inat
e (+
).
WA
KA
MA
TSU
et
al.,
200
6
anti-
pep
tide
vira
l N
pro
tein
/ S
PF
Bel
tsvi
lle w
hite
Tur
key
and
com
mer
cial
M
ediu
m
Whi
te to
m tu
rkey
s
Con
junt
ival
sa
c/
VV
=Cal
iforn
ia
1083
, V
N=T
urke
y N
D,
M=
Roa
kin,
L=L
aSot
a, A
=Iow
a 15
19
Spl
een
(VV
+, V
N+)
, th
ymus
(V
V+)
, bu
rsa
of F
abric
ius
(VV
+, V
N+)
, ey
elid
(V
V+,
VN
+),
Har
deria
n gl
and
(VV
+,
VN
+,
A+)
, ph
aryn
x (V
V+,
V
N+)
, cr
op
(VV
+,
VN
+),
esop
hagu
s (V
V+,
VN
+, M
+), p
rove
ntic
ulus
(V
V+,
VN
+),
panc
reas
(VV
+, V
N+,
A+)
, sm
all
inte
stin
e (V
V+,
VN
+),
ceca
l to
nsils
(V
V+,
V
N+,
A
+),
larg
e in
test
ine
(VV
+,
VN
+), c
auda
l tho
raci
c ai
r sa
c (V
V+, A
+), t
rach
ea (
VV
+,
M+,
L+,
A+)
, lu
ng (
VV
+, V
N+,
A+)
, he
art
(VV
+, V
N+,
M
+, A
+),
liver
(V
V+)
, ki
dney
(V
V+,
VN
+),
brai
n (V
V+,
V
N+)
and
bon
e m
arro
w (V
V+)
.
PIA
CE
NTI
et
al
., 20
06
Deg
ree
of v
irule
nce:
VV
= ve
loge
nic
visc
erot
ropi
c, V
N=
velo
geni
c ne
urot
ropi
c, M
= m
esog
enic
, L=
lent
ogen
ic, A
= as
ympt
omat
ic.
Oth
er: +
= p
ositi
ve re
actio
n, -
= ne
gativ
e re
actio
n.
39
3. Newcastle Disease Virus (NDV)
3.1. General considerations
3.1.1. Virus characteristics
In current virus taxonomy, NDV is classified in the genus Avulavirus, sub-family
Paramymovirinae, family Paramyxoviridae, order Mononegavirales (KNIPE et al.,
2007; ALEXANDER and JONES, 2008).
This virus contains non-segmented single-stranded RNA genomes of negative
polarity (EMMERSON, 1999; KNIPE et al., 2007) and replicates entirely in the
cytoplasm (KNIPE et al., 2007). A lipid envelope contains two surface glycoproteins,
which surround the virion, Hemagglutinin- Neuraminidase (HN) and Fusion (F). The
molecules are considered as most important in the attachment and fusion of the virus
with the host cell (see chapter 3.2). Inside the envelope lies a helical nucleocapsid
core containing the RNA genome and the nucleocapsid (N), phospho- (P) and large
(L) proteins, which initiate intracellular virus replication. Residing between the
envelope and the core is the viral matrix (M) protein that is important in virion
architecture (KNIPE 2007, Fig. 3).
The L protein and the P protein are responsible for viral synthesis. The M protein
interacts with both the viral membrane and the nucleocapsid and is involved in viral
assembly. N binds tightly to the genomic and antigenomic RNAs, producing helical
structures. RNA that is not bound to NP cannot be transcribed or replicated; the
concentration of free N within the infected cell is thought to control the relative rates
of transcription and replication (EMMERSON, 1999).
40
Figure 3. Viral proteins of NDV (http://wordsandwar.com, modified) HN= Hemmaglutinin-neuraminidase protein, F= Fusion protein,N= Nucleocapsid protein, L= large protein and M= Matrix protein.
NDV have been grouped into five pathotypes that are related to the disease signs
produced in infected fully susceptible chickens: 1) viscerotropic velogenic, 2)
neurotropic velogenic, 3) mesogenic, 4) lentogenic and 5) asymptomatic enteric.
Three of the most common tests used to determine the virus pathogenicity: 1) Mean
Death Time (MDT) in eggs, 2) Intracerebral Pathogenicity Index (ICPI) in 1 day old
chicken and 3) Intravenous Pathogenicity Index (IVPI) in 6 week old chicken (see
Table 4, ALEXANDER 1998).
41
Table 4. Pathotypes and pathogenicity indices of NDV (ALEXANDER, 1998)
Pathotype Range of indices Examples of
MDT ICPI IVPI viruses
Viscerotropic velogenic � 60 hrs 1.5 - 2.0 2.0 - 3.0 Herts´33
Neurotropic velogenic � 60 hrs 1.5 - 2.0 2.0 - 3.0 Texas GB
Mesogenic 60-90 hrs 1.0 - 1.5 0.0 - 0.5 Komarov
Lentogenic � 90 hrs 0.2 - 0.5 0.0 Hitchner B1
Asymptomatic � 90 hrs 0.0 - 0.2 0.0 Ulster 2C
MDT = Mean Death Time, ICPI = Intracerebral Pathogenicity Index, IVPI = Intravenous Pathogenicity Index.
3.1.2. Epidemiology and geographic distribution
The virus is distributed almost worldwide (EMERSON, 1999; ALDOUS and
ALEXANDER, 2001), and is defined as a list A disease (obligatory denounce) by the
Office International de Epizooties (OIE). Depending of the pathotype, it may cause
economic losses in domestic poultry, especially in chickens (KALETA, 1992).
Mildly virulent NDV strains are endemic and circulate in many poultry populations.
They are thought to impair clearance of other respiratory pathogens, leading to
secondary infections that cause disease (NAKAMURA et al., 1994).
3.1.3. Prevention and control
Full protection against NDV requires a combination of hygienic precautions and
vaccination. Vaccines available against NDV are either live strains of low virulence or
inactivated strains. Among live avirulent strains, Hitchner B1 and La Sota are used
extensively worldwide as primary vaccines. Vaccines can be administrated via
drinking water, aerosol, individual application to the eye or nostril, beak dipping or
injection (EMMERSON, 1999).
42
Many countries impose quarantine on birds on importation, which is aimed
specifically at the detection and elimination of birds infected with NDV. Generally, the
requirement is isolation for a period of at last 35 days, with close veterinary
supervision and virus isolation monitoring for NDV (JORDAN et al., 2001).
3.1.4. Diagnostic
A rapid diagnosis and characterization of NDV and virulence assessment is important
to reduce its impact in a poultry farm. For virus isolation from dead birds the samples
should consist of oro-nasal or cloacal swabs, lung, kidneys, intestine, spleen, brain,
liver and heart tissues (OIE, 2004; JORDAN et al., 2001). From alive birds both
tracheal and cloacal swabs and odder fresh feces should be included, for a
serological test a sample of serum is used (OIE, 2004).
Virus isolation is realized by virus culture, virus identification, pathogenicity indices
(OIE, 2004) and molecular based techniques (CREELAN et al., 2002; ALDOUS et
al., 2003; OIE, 2004; PHAM et al, 2005). For example, GOHM et al. (2000) employed
RT-PCR technique to detect NDV in conjunctiva, lung, caecal tonsil, kidney and
feces of infected chickens with 4 and 6 days post infection. An other study,
conducted by PHAM et al. (2005), demonstrates the use of a Loop-Mediated
isothermal amplification to diagnose the presence of NDV in culture of isolated and
clinical samples. This technique was as sensitive and as specific as the nested PCR
employed.
A hybridization of PCR fragments with fluorogenic probes specific for the pathotype
using a modified TaqMan procedure was used for estimating the pathogenicity of
NDV. On the basis of the results obtained, the authors suggest that this protocol
allows determining the virulence of most ND isolates rapidly and reproducibly
(ALDOUS et al., 2001).
43
A serological diagnostics of NDV can be obtained by different tests, for example
neutralization, enzyme-linked immunosorbent assay (ELISA), hemmaglutination test
(HA), hemmagglutination inhibition test (HI); the HI is most widely used (OIE, 2004).
4.1.5 Incubation period, transmission routes and host range
The NDV has an incubation period from 3 to 28 days depending on the virulence of
the virus and the species affected. In chickens, this is 2 to 15 days (average 5 days)
(RITCHIE, 1995). The virus is highly contagious (KALETA, 1992) and can be
transmitted by inhalation and/or ingestion (EMMERSON, 1999). The mode of
transmission from bird to bird is dependent on the organs in which the virus
multiplies. Birds showing respiratory disease presumably shed virus in aerosols of
mucus, which may be inhaled by susceptible birds.
Viruses that are mainly restricted to intestinal replication may be transferred by
ingestion of contaminated feces, either directly or in contaminated food or water, or
by inhalation of small infectious particles produced from dried feces. Viruses
transmitted by the respiratory route in a community of closely situated birds may
spread with alarming rapidity (JORDAN et al., 2001).
Humans seem to play the central role in the spread of NDV, usually by the movement
of live birds, fomites, personnel and poultry products from affected premises to
susceptible birds. Feral birds and other wildlife have undoubtedly contributed to the
spread of disease during epizootics, either by infection or by mechanical transfer, but
their exact role has not been fully evaluated (JORDAN et al., 2001).
NDV has been shown to infect most species of birds and many species of mammals,
including the human (RITCHIE, 1995). Over 250 species of birds have been reported
to be susceptible to natural or experimental infection by NDV (ALEXANDER and
JORDAN, 2008), both domestics and wildlife birds (OIE 2004). The infections have
44
been documented in Anseriformes, Columbiformes, Psittaciformes, Passeriformes,
Falconiformes, Cuculiformes, Strigiformes, Sphenisciformes, Gruiformes, Piciformes,
Phasianidae, Struthioniformes and Pelicaniformes (RITCHIE, 1995).
The chicken is the domestic bird specie, which is most affected (EMMERSON, 1999;
OIE 2004; ALEXANDER and JONES, 2008), while ducks and geese appear to be
resistant (EMMERSON, 1999; ALEXANDER and JONES, 2008). Canaries, parrots
and cranes have also been reported to be fairly resistant to infection. In psittacine,
pigeons and cormorants the disease may occur chronically and could be introduced
or erupt at any moment (BROWN et al., 1999).
3.1.6. Clinical signs
The animals can show different clinical signs, which depend on factors such as:
virulence of the virus, host species, age of host, infections with other organisms,
environmental stress and immune status (JORDAN et al., 2001; OIE 2004). They can
vary from asymptomatic enteric infections to systemic infections causing 100%
mortality (BEARD and HANSON, 1984).
The first clinical sign in birds is loss of appetite, usually about the fourth or fifth day
after infection. Respiratory rate is increased and characterized by long, gasping
inhalation through a half-opened beak. In many cases, there is diarrhea and thick
mucus discharge from the nostrils and the mouth. The bird’s body temperature rises
gradually until about the sixth or seventh day and falls quickly to below normal just
before death (EMMERSON, 1999).
Experimentally infected animals show hyperemia and edema of the conjunctiva,
cyanosis of the comb, depression and diarrhea between 3 and 15 days p.i. (GOHM
et al., 2000). BROWN et al. (1999) reported the following clinical signs in chickens
inoculated with different strains of the NDV: Infected with a viscerotropic strain, the
45
birds are severely depressed, show marked bilateral conjunctivitis, lateral
recumbency, and are unable to right themselves up and finally die. Animals
inoculated with mesogenic and lentogenic strains do not demonstrate signs of clinical
disease.
The highly virulent viruses (velogenic) may produce peracute infections. The
chickens can show depression, prostration, diarrhea, edema of the head (JORDAN
et al., 2001). Nervous signs may also occur, with mortality reaching 100% (JORDAN
et al., 2001; OIE, 2004).
Mesogenic viruses usually cause severe respiratory disease, followed by nervous
signs, with mortality up to 50% or more. The lentogenic viruses may cause either no
clinical disease or respiratory disease; in broilers, it has been associated with
multiple infections of the respiratory tract (JORDAN et al., 2001), also a subclinical
enteric infection can be observed (OIE, 2004).
NDV may cause mild conjunctivitis in humans (RITCHIE, 1995; EMMERSON, 1999)
and generalized malasia (RITCHIE, 1995).
3.1.7. Histopathologic changes observed in chicken organs infected with NDV
Velogenic viscerotropic pathotype: Largely devoid of mononuclear cells, extensive
deposits of fibrin replacing periarteriolar lymphoid sheaths in spleen, and mild
lymphoid depletion of bursa, thymus, spleen and cecal tonsil are reported (BROWN
et al., 1999; WAKAMATSU et al., 2006).
Nevertheless, massive destruction of intestinal lymphoid areas, most prominent in the
cecal tonsil, ulceration of the intestinal epithelium, as well as disruption of cardiac
myofibers and accumulation of macrophages within the myocardium are observed
(BROWN et al., 1999), or focal neuronal degeneration and gliosis in the brain
46
(BROWN et al., 1999; WAKAMATSU et al., 2006). Additionally, hemorrhagic lesions
of the digestive tract (GOHM et al, 2000), particularly the proventriculus (JORDAN et
al., 2001) and necrotizing laryngitis (WAKAMATSU et al., 2006) are reported.
Velogenic neurotropic: Perivascular cuffing, neuronal degeneration, modest
lymphoid depletion in the spleen and variable disruption of heart muscle with
inflammation may occur (BROWN et al., 1999).
Mesogenic: Splenic lymphoid hyperplasia, myocardial inflammation with
degeneration of myofibers and infiltration of macrophages are reported (BROWN et
al., 1999).
Lentogenic: Splenic lymphoid hyperplasia and prominent lymphoid follicles in air sac
occur (BROWN et al., 1999). In trachea hyperemia (GOHM et al, 2000), excessive
catarrhal exudates or severe hemorrhages (EMMERSON, 1999) can be observed.
3.2 Pathogenesis
3.2.1 Recognition and fusion mechanism
Viral infection is initiated by a collision between the virus particle and the cell.
However, a virus is not able to infect every cell it encounters, it must come in contact
with the cell and tissue in which it can replicated. Such cells are normally recognized
by means of a specific virion-cell surfaced receptor interaction (FLINT et al., 2004).
Enveloped viruses enter cells by fusing of the viral envelope with the cell membrane
(WANING et al., 2004).
For all paramyxoviruses, coexpression of F and HN proteins is required for fusion. It
was hypothesized that a type-specific interaction would occur between these
proteins. One of the difficulties in the work studying HN is that mutations often affect
47
more than one of the three know biological activities: 1) it recognizes sialic acid-
containing receptors on cell surfaces, 2) it promotes the fusion activity of the F protein,
thereby allowing the virus to penetrate the cell surface, and 3) it acts as a
neuraminidase, removing sialic acid from progeny virus particles to prevent viral self-
agglutination (CRENNELL et al., 2000; KNIPE et al., 2007).
Activation of most paramyxovirus F proteins occurs at neutral pH. And is thought to
be triggered by steps including (i) binding of the viral attachment protein (HN or H) to
its cell surface receptor, (ii) HN interacting with F, and (iii) the HN/F interaction
leading to changes in F that mediate membrane fusion (CRENNELL et al., 2000;
RUSSELL et al., 2001).
In addition, HN mediates enzymatic cleavage of sialic acid (neuraminidase activity)
from the surface of virions and the surface of infected cells. HN is glycosylated and
contains four to six potential sites for the addition of N-linked carbohydrate chains
(KNIPE et al., 2007).
Upon adsorption of the virus to the cellular receptor, the viral membrane fusion with
the cellular plasma membrane is occurring at a neutral pH, which is found at the cell
surface; consequently, the helical nucleocapsid is released into the cytoplasm. All
aspects of the viral replication occur in the cytoplasm, as also in the Influenza virus.
A scheme of the life cycle of the Influenza virus is shown in Fig. 4 (KNIPE et al.,
2007).
48
Figure 4. Life cycle of Influenza virus (http://www.avianflu.umd.edu, modified) Viral proteins= PB1, PB2, PA, NP, M1, NS1, NS2, HA, NA and M2.
3.2.2 Virulence
Different theories have been proposed regarding the factors that determine the
virulence of the NDV:
(1) HN protein contributes to the virulence of NDV. Viral tropism in a susceptible
host could often be determined by virus-receptor interactions
HN is an integral membrane glycoprotein with a single transmembrane domain
having a terminal globular head in which its hemoadsorption capacity resides
(VARGHESE et al., 1983; CRENNELL et al., 2000). This protein is responsible for
49
the attachment of the virus to the sialic acid containing receptor on the cell surface
(NAGAI 1993; EMMERSON, 1999; CONNARIS et al., 2002) and for prevention of
self-aggregation of viral particles during budding (EMMERSON, 1999; LAMB and
KOLAKOFSKY, 2001; CONNARIS et al., 2002).
Analysis of the three-dimensional structure of Newcastle disease virus (NDV) HN
protein revealed the presence of a large pocket, which mediates both receptor
binding and NA activities. All paramyxovirus HN proteins are pairs of dimers. The
stalk supports a terminal globular domain in which attachment and NA activities
reside and in which all the antigenic sites were present (MIRZA, 1993). In the case of
NDV, 95% of HN purified from virions under nonreducing conditions consists of
homodimers. In some strains of NDV, disulfide-linked dimmers are formed via Cys 123
(CRENNELL et al., 2000).
Within the last decade very import information were obtained concerning the three-
dimensional structure of NDV. CRENNELL et al. (2000) reported the crystal structure of
the globular head region of HN from the Kansas strain of NDV. The structure derived
using multiple heavy atom isomorphous replacement, followed by multicrystal
averaging, noncrystallographic symmetry averaging and phase extension that exploited
the severe nonisomorphism observed in the crystals. A ligand-free structure was
resolved to 0.25 nm from an orthorhombic crystal form grown at pH 4.6. A complex
with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en, DANA)
was resolved to 0.28 nm, from a hexagonal crystal form obtained at pH 6.5 by
cocrystallization, in which the NA active site was clearly identified. The pH 4.6
orthorhombic crystal form was soaked with sialyllactose (Neu5Ac (2,3)Gal (1,4)Glc)
to provide a complex that was resolved to 0.2 nm that revealed the -anomer of sialic
acid bound alone in the active site.
HN protein has been postulated as a primary molecular determinant of virulence
(NAGAI et al., 1976; GLICKMAN et al., 1988). Most mutations around the binding site
50
result in loss of neuraminidase activity, whereas the effect on receptor binding is
more variable (CONNARIS et al., 2002; FERREIRA et al., 2004).
In order to determine the role of the HN protein in NDV virulence, reverse genetics
procedures were used. Genes of a virulent recombinant NDV strain (rBeaudette C)
and an avirulent recombinant NDV strain (rLaSota) were exchanged, and a
significant difference from those of their parental strains was observed. The tissue
tropism of the viruses was shown to be dependent on the origin of the HN protein.
The chimeric virus with the HN protein derived from the virulent virus exhibit a tissue
predilection similar to that of the virulent virus and vice versa. These results were
consistent with the hypothesis that the virulence of NDV is mutagenic, and that the
cleavability of F protein alone does not determine the virulence of a strain (HUANG et
al., 2004).
Mutations were generated in residues at the putative catalytic site of the haemagglutinin-
neuraminidase (HN) protein of NDV Clone 30 strain (Arg498, Glu258, Tyr262, Tyr317
and Ser418), and their effects on its three associated activities were studied. Expression
of the mutant proteins at the surface of HeLa cells was similar to that of the wild-type.
Sialidase, receptor-binding and fusion-promotion activities were affected to different
degrees for all mutants studied. Mutant Arg498Lys lost most of its sialidase activity,
although it retained most of the receptor-binding activity, suggesting for the former
activity, that besides the presence of a basic residue, the proximity to the substrate
molecule is also important to their activity, as Lys is shorter than Arg.
However, a kinetic and thermodynamic study of the sialidase activity of the Tyr262Ser
and Ser418Ala mutants was performed and revealed that the hydroxyl group of these
residues also plays an important role in catalysis, since such activity was much less
effective than that of the wild-type, and these mutations modified their activation energy
for sialidase catalysis. The discrepancy of the modifications in sialidase and receptor-
binding activities in the mutants analysed does not account for the topological
51
coincidence of the two sites. These results also suggest that the globular head of HN
protein may play a role in fusion-promotion activity (FERREIRA et al., 2004).
Recently, a second sialic acid binding site on HN was revealed, a thiosialoside
Neu5Ac-2-S-(2,6)Gal1OMe structure, suggesting that NDV HN contains an additional
sialic acid binding site (BOUSSE et al., 2004; ZAITSEV et al., 2004), but the biological
importance of the second sialic acid-binding site is unclear (KNIPE et al., 2007).
To evaluate the role of the second binding site on the life cycle of NDV, rescued mutant
viruses whose HNs were mutated at Arg516, a key residue that is involved in the
second binding site, were used. Loss of the second binding site on mutant HNs was
confirmed by the hemagglutination inhibition test, which uses an inhibitor designed to
block the NA active site. Characterization of the biological activities of HN showed that
the mutation at Arg516 had no effect on NA activity. However, the fusion promotion
activity of HN was substantially reduced by the mutation. Furthermore, the mutations
at Arg516 slowed the growth rate of virus in tissue culture cells. These results suggest
that the second binding site facilitates virus infection and growth by enhancing the
fusion promotion activity of the HN (BOUSSE et al., 2004).
(2) Amino acid sequence surrounding the F protein cleavage site. Host proteases
that cleave the F protein of virulent strains are present in more tissues than
those that cleave the F protein of non-virulent strains
The virulence of NDV is associated with differences in the amino acid sequence
surrounding the post translational cleavage site of the F0 protein (BALLAGI-
PORDANY et al., 1996; ALEXANDER, 2000).
The F protein is produced as a precursor molecule (F0), which must be proteolytically
cleaved, producing two disulfide-linked polypeptides, F1 and F2 (SCHEID et al., 1974;
NAGAI et al., 1976; EMMERSON, 1999). The cleavage activation of F0 is catalyzed
52
by host cell proteases with trypsin-like activity. Later in infection, the F proteins
expressed at the plasma membrane of infected cells can mediate fusion with
neighboring cells to form syncytia and giant cells (KNIPE et al., 2007).
These active proteins permit the penetration of the virus into the host cell plasma
membrane (GLICKMAN et al., 1988; EMMERSON, 1999), and this fusion event
occurs at neutral pH (KNIPE et al., 2007). The amino terminus of F1 is extremely
hydrophobic and has functions as the insertion peptide, promoting fusion of the viral
and cellular envelopes (RICHARDSON et al., 1980).
MORRISON et al. (1991) conducted a study to determine the role of F protein in the
membrane fusion. In this study, the cDNA derived from the fusion gene of the virulent
AV strain of NDV was expressed in chicken embryo cells by using a retrovirus
receptor. The F protein expressed in this system was transported to the cell surface
and cleaved into the disulfide-linked F1-F2 form found in infectious virions. According
to the results, the authors concluded that the F protein and the HN protein are
required for membrane fusion, and that the presence of other viral attachment
glycoproteins expressed in the same cell will not substitute for the HN protein.
Some authors proposed a mechanism of viral tropism in which host cell proteases
activate the F glycoproteins. The proteases cleave the biologically inactive single-
chain precursor F0 into the active, disulfide-bonded two-chain molecule F with the
NH2-terminal F2 and COOH-terminal F1 subunits; this cleavage is a precondition for
viral infectivity. The F0 of virulent NDV strains is activated by ubiquitous proteases
and the infection is systemic, while avirulent strains undergo F0 cleavage only in a
few limited tissue types, hence causing an infection localized in particular organs with
trypsin-like enzymes, such as the respiratory and alimentary tract (NAGAI et al.,
1989; OGASAWARA et al., 1992; PEETERS et al., 1999).
In the virulent isolates, the F1 always consists of four basic amino acids with an
intervening glutamine residue (Arg-Arg-Gln-Arg/Lys -Arg). In contrast, in the five
53
avirulent isolates examined here, a neutral amino acid, glycine, is found in place of
the basic arginine residues at position 1 and 4 of the pentapeptide (Gly-Arg-Gln-Gly-
Arg). These suggest that a high content of basic residues in the cleavage site may be
at least one of the requirements for NDV to form plaques in most cell types
(GLICKMAN et al., 1988).
(3) Other proteins may play an important role in the NDV virulence
PHAM et al., 2004 consider that the M protein can play an important role regarding
the virulence of NDV because the virus is losing some capside proteins when release
the host cell. In the virus particle, the M protein shell is believed to make numerous
contacts with the nucleocapsid (KNIPE et al., 2007).
4 Aims
The aim of the present study is to determine the significance of glycosylation
patterns, in the context of virus-cell interaction, and possible alterations in infected
tissues. For this purpose, we used a panel of lectins for characterization of the
normal distribution of terminal sugar moieties in different organs of healthy uninfected
chicken embryos in comparison to infected ones. NDV of different pathotypes
(lentogenic and mesogenic) were selected as an infectious model agent. Additionally,
histopathology (H&E and Pappenheim´s staining), presence of mucins (TB),
presence of glycoconjugates (AB-PAS) and confirm the presence of the virus (IHC)
was performed in embryonic tissues. To our knowledge, this is the first report with
NDV regarding these questions.
54
III. Material and Methods
1 Introductory remarks
The use of embryonated eggs was justified considering the following facts:
(1) NDV is efficiently replicating in embryonated eggs. Therefore, they are used
as a diagnostic tool for the determination of NDV pathogenicity.
(2) The use of embryonated eggs reduces the costs of maintenance and,
according to the definitions derived from the German animal-protection
regulations, the number of animal experiments.
The following selection criteria for histological evaluation were used:
(1) The embryonated eggs were SPF (see Annex 1 for details) and had no
maternal antibodies against NDV, and therefore no interference with the virus
infection.
(2) Embryonated eggs with a minimum of 4 days p.i. were used, because the
incubation time of the virus is 4-5 days (in average). They were kept alive
during this period.
(3) Only embryonated eggs which were antigen-positive (after HA-test) were
selected for histological examination.
55
Therefore, it was necessary to standardize the embryonated egg infection model; the
procedure is described in chapter 2.
2 Determination of the LD50 of NDV
The activity of a virus in a biological suspension is measured quantitatively by
procedures that consist of preparing dilutions of the suspension and determine the
dilution at which a particular biological activity is still detectable (VILLEGAS, 1998).
For this purpose, quantitative assays in fibroblasts and chicken embryos were
performed to determine the LD50 of the NDV pathotype employed, using the method
of REED and MUENCH as described by VILLEGAS (1998).
To perform quantitative assays, serial dilutions of the virus were inoculated in the
host and after variable incubation times, at each dilution, the hosts were evaluated
and the virus effect was determined to be either positive or negative. In cell culture
presence of CPE were consider as positive and in the embryonated chicken eggs
died embryo were considerate as positive (more details chapter 2.1 and 2.2). The
proportion of the positive hosts was recorded and used to calculate the concentration
or titer of the viral suspension.
2.1 Virus titration in cell culture (chicken fibroblasts)
Chicken embryo fibroblasts were grown in 3 cluster plates containing 96 wells with a
concentration of 7.5X1015 cells/ml. They were incubated at 37.1°C in an atmosphere
of 6.2% CO2. The Leibovitz/McCoy´s maintenance medium was used and 1% FBS.
56
When the monolayers were confluent, each plate was infected with 200 μl of a virus
suspension with a serial dilution of 1:10 to 1:1011 of different NDV pathotypes: Herts
33 (velogenic), Komarov (mesogenic) and HB-1 (lentogenic). The wells were
incubated with the virus suspension for 1 hour at 37.1°C, and then washed carefully
with PBS to eliminate dead cells and debris. Afterwards, fresh maintenance medium
was added.
The monolayer was observed daily with an inverted microscope (Reichert-Jung
Biostar®) until cellular morphological alterations, called CPE, were present (maximal
7 days). For HB-1, incubation of the fibroblasts with trypsin was required, before
inoculation, because the lentogenic pathotype of the NDV infects tissue with trypsin-
like proteases (100 μl, concentration 0.02%, 30 minutes).
The presence of CPE was recorded to determine the LD50, by the method of REED
and MUENCH (VILLEGAS, 1998). The result was used to prepare infected fibroblast
as a positive control for the IHC (chapter 5.1.4). Virus titration using the original NDV
stock and allantoic fluid of the infected embryonated eggs were performed to
ascertain virus replication and to determine the LD50.
To determine the LD50, the following equation was used (VILLEGAS, 1998):
PD= [Percentage infected at dilution next above 50%] - 50%
Percentage infected at dilution - Percentage infected at dilution
next above 50% next below 50%
log of the 50% endpoint= log dilution above 50%- (PD x log dilution factor)
The endpoint dilution has no units, and the titer is defined as the number of infectious
units per unit volume. Therefore, the titer of the preparation is the negative
exponential of the endpoint dilution and is expressed as LD50/dose.
57
2.2 Inoculation of embryonated chicken eggs with NDV
2.2.1 First viral passage
68 embryonated eggs from SPF chickens Valo (Lohmann®) were inoculated via the
allantoic sac at 11th incubation days (i.d.) with serial dilutions of NDV (1:105 - 1:1011):
20 were inoculated with NDV HB-1, 24 with Komarov and 24 with Herts 33
pathotypes. Two eggs were inoculated as negative controls with BME.
Before inoculation, all eggs were candled for viability of the embryos. For inoculation
the eggs were placed on an egg flat, with their air cell on the top, and the area
directly at the top was disinfected with alcohol. Then a small hole was drilled through
the eggshell along the center axis at the top of the egg. Each egg was identified with
a number and using a tuberculin syringe 0.1 ml solution was inoculated by inserting
the needle and injecting the desired amount. The hole was sealed with the adhesive
Uhu-hart®. The eggs were then placed in the incubator at 37 ºC and candled two
times a day for 8 days.
The time p.i. at which the embryos were dead was recorded to determine the LD50 as
described previously (see chapter 2.1) or the survival time of the embryos;
respectively. Embryos, which died the day after inoculation, were discarded, because
during the first 18-24 hours p.i. nonspecific deaths usually result from the
manipulation of embryonated chicken eggs (VILLEGAS, 1998).
From each dead embryonated chicken egg allantoic fluid was collected. To collect
allantoic fluid, the shell over the air cell was disinfected, cracked and removed with
forceps. With a second forceps the eggshell was removed. Using a 5 ml pipette, the
allantoic fluid was aspirated and placed into a sterile tube, then stored at -20ºC.
58
Allantoic fluid was processed for HA-test, with the following protocol:
(1) First 25 μl of PBS was dispensed into each well of a plastic V-bottomed
microtitre plate.
(2) Subsequently 25 μl of the allantoic fluid were placed in the first well and then
the serial dilutions were made.
(3) Then 25 μl of 1% (v/v) chicken RBCs was dispensed to each well. This
suspension was then mixed by shaking the plate gently, and was incubated 30
min at room temperature. HA was determined by tilting the plate and
observing the presence of tear-shaped streaming of the RBCs.
2.2.2 Second viral passage
A second virus passage was necessary because, depending on the virus
pathogenicity, the embryos died early. Therefore, the rational for conducting a
second passage experiment was to attenuate the ND stock virus, thus reducing the
mortality and redoing the surviving embryos as useful for the following studies. Serial
dilutions of allantoic fluid of the first passage were inoculated via allantoic sac, as
described in chapter 2.2.1, at 11th i.d. 24 embryos were inoculated with each of the
three pathotypes, and four were inoculated with BME as a negative control. The
embryos were observed as described in chapter 2.2.1.
59
3 Cytological studies
Infected (lentogenic, mesogenic and velogenic) and uninfected chicken embryo
fibroblasts were microscopically investigated employing IHC as described in chapter
5.1.4.
Four bottles for culture cells with 10 ml chicken embryo fibroblasts suspension in
BEM medium were incubated at 37°C and 5.2% CO2 until a complete monolayer was
observed.
Following, 3 bottles were infected with different NDV pathotypes (4.5 ml BEM, 0.5 ml
trypsin and 25 μl virus – NDV-Herts 33, NDV-Komarov and HB-1 respectively), and
one was used as negative control (4.5 ml BEM and 0.5 ml trypsin); they were
incubated for 30 min at 37°C and 5.2% CO2.
The fibroblasts were washed with PBS and incubated (37°C, 5.2% CO2), and
observed until the CPE was present (after 3 to 5 days).
Subsequently, the fibroblasts were washed twice with DBP 1X, 2 ml Trypsin were
added, and incubated for 5 min (37°C, 5.2% CO2); then 100 μl FBS (Biochrom AG®)
was added. Hereafter medium with fibroblasts was collected in a tube and
centrifuged (Sigma 4-10®) 5 min at 1000 RPM, the liquid was decanted and 3 ml DBP
1X was added to each tube, and the pellet diluted.
A volume of 100 μl of the solution was transferred into a sample centrifugation
chamber of the Cytospin centrifuge (Shandon®), and centrifuged at 3500 U/5 min. In
this way, the fibroblasts were sedimented on a microscopy slide (HistoBond,
Marienfeld®).
60
They were fixed with Bouin’s solution (BÖCK, 1989) for 48 hours. Bouin’s solution
was prepared as follows:
(1) 1500 ml Picric acid (Annex 2).
(2) 500 ml Formaldehyde (37%).
(3) The solution was filtered.
(4) At day of use 5 ml glacial acid acetic / 100 ml was added.
Subsequently, they were embedded in paraffin wax (LILLIE and FULLMER, 1976),
as follows:
(1) The samples were first placed for 24 hours in 70% ethanol.
(2) Then for 24 hours in 80% ethanol.
(3) Followed by 2 hours in 90% ethanol, 100% ethanol, isopropanol and
xylene, respectively.
(4) Then kept overnight at 60°C in paraffin (Paraplast, Sherwood®).
(5) Finally, the samples were transferred to fresh paraffin, incubated for 2
hours. This procedure was repeated once in order to decrease xylene
concentrations.
4 Studies of chicken embryos
Histological observations were performed to compare the glycosylation patterns of
uninfected chicken embryos and those infected with NDV (lentogenic and
mesogenic) at different days of incubation. Since it was observed during the
experiments that the velogenic pathotype (NDV-Herts 33) induced earlier mortality
(� 4 days p.i.) than the two other pathotypes (NDV-Komarov and HB-1), despite a
61
second passage to reduce mortality, this NDV pathotype was excluded from further
experimentation (section 2.2.2).
4.1 Inoculation of chicken embryos
Two groups were used:
(1) Control group: Fifty-five embryonated SPF chicken eggs were taken after
11th, 13th, 15th, 17th and 19th i.d., in order to compare the glycosylation
pattern at the inoculation time and the end point.
(2) Infected group: This group comprised 220 embryonated SPF chicken eggs.
They were inoculated via the allantoic sac, as described in the chapter 2,
with different NDV pathotypes. Subgroups were formed as described in
Table 5.
Table 5. Subgroups of embryonated chicken eggs inoculated
Number of embryos NDV Pathotype/ doses Day of incubation
40 HB-1/1010LD50 11
40 Komarov/109,3LD50 11
60 HB-1/1010 LD50 15
60 Komarov/109,3LD50 15
10 BME 11
10 BME 15
The embryos were euthanized by snap freezing, four days p.i. From each egg,
allantoic fluid was taken for further virological analysis, beginning with the HA-test
(described in the chapter 2.2.1). From the infected embryos, only those that were
positive in the HA-test were used for the histological studies (chapter 2.2.1). A titer
over 1:8 was considered positive. To confirm the presence of NDV, HI-test
62
(STEPHAN and BEARD, 1998) was performed with a polyclonal chicken anti-serum
against NDV (in house made, Clinic for Poultry, University of Veterinary Medicine
Hannover).
Once euthanized, the embryos were placed in 250 ml Bouin’s solution (BÖCK, 1989)
for 48 hours to fix the tissues (preparation of Bouin’s solution was described in
chapter 3).
4.2 Embedding
4.2.1 Paraffin wax embedding
Fifty embryos of the group 1 and 45 of the group 2 (positive to HA-Test) were cut in
six pieces (transversal sections at sex blocks, Fig. 5) and embedded in paraffin wax
(as described in chapter 3) and finally paraffin blocks were prepared.
Sections were cut with a microtome (Leitz® 1512) at 5 μm thickness. The sections
were deparaffinised in xylene and hydrated through a series of graded alcohol, as
follows:
(1) 3 min in isopropanol,
(2) 3 min in ethanol 96%
(3) 3 min in ethanol 80%
(4) 3 min in ethanol 70%
(5) 2 min in distilled water
They were stained with H&E and Pappenheim´s staining for normal histology, AB-
PAS to determine the presence of glycoconjugates, IHC to detect NDV antigen and
LH to determine the glycosylation pattern of the embryo tissues (procedure, see
chapter 5).
63
Fig 5. Localization of sections across the embryo (250X) 1-6: Section levels made in each chicken embryo.Organs relevant four our investigation are designated (a-l) in each section: a. esophagus, b. trachea, c. thymus, d. lung, e. liver, f. proventriculus, g. spleen, h. air sac, I. kidney, j. ventriculus, k. intestine, l. bursa of Fabricius.
64
4.2.2 Technovit 7100 embedding
Trachea, lung, esophagus, small intestine, large intestine, thymus, liver, spleen, and
bursa of Fabricius from a total of 5 embryos of group 1 and 14 of group 2 were
embedded in Technovit 7100 (Kit from Heraeus-Kulzer®), as follows (HANSTEDE
and GERRITIS, 1983):
(1) The organs were placed in 70% ethanol with 1 drop Ammonia solution 25%
(Merck®). The ethanol was repeatedly changed until the yellow color was
absent.
(2) Then the samples were placed for 24 hours in 80% ethanol,
(3) 20 min in 90% ethanol,
(4) 20 min in 96% ethanol,
(5) Overnight in Technovit 7100 Solution I (100 ml Technovit 7100 and 1 g
Hardener I).
(6) Finally, the blocks were prepared in the Technovit 7100 Solution II (15 ml
Technovit 7100 Solution I and 1ml Hardener II).
(7) Incubation overnight in a stove for hardening.
Sections were cut with a motor-driven microtome (Reichert-Jung® 1114/Autocut) at 3
μm thickness, and stained with H&E for normal histology and TB to determine the
presence of mucins (procedure, see chapter 5).
65
5 Histological staining
5.1 Paraffin sections
5.1.1 Hematoxylin and Eosin
For a good differentiation of nuclei and cytoplasm, the preferred method is H&E
staining. With this staining nuclei are blue and cytoplasm is pink, the nuclear and cell
boundaries are well defined (LILLIE and FULLMER, 1976).
The paraffin slides were stained with the following protocol (BÖCK, 1989):
(1) 8 min hematoxylin (Delafield formula) (Annex 3)
(2) 10 – 15 sec in 0,1 % HCl in distilled water
(3) 15 min in water
(4) 5 min in eosin (Annex 4) in distilled water with 5 drop acetic acid glacial
(Riedel-de Haën®)
(5) 2 min 70% ethanol
(6) 2 min 80% ethanol
(7) 2 min 96% ethanol
(8) 2 min isopropanol
(9) twice 5 min xylene
(10) Mounted with Eukitt (Kindler®)
5.1.2 Alcian Blue – PAS, pH 2.5
Alcian blue and PAS are common stains used in combination for the histochemical
detection of mucosubstances. This method allows the observation of complex neutral
(red) and acidic (blue) glycoconjugates. It is not possible to differentiate carboxyl and
66
sulphated groups. And in case of a mixed reaction a violet color is observed
(PEARSE 1972).
Paraffin slides were stained with the following procedure (PEARSE, 1972):
(1) 60 min alcian blue 8GX, Sigma®.
(2) Washed in distilled water
(3) 10 min 0.8% periodic acid
(4) 3 times 3 min in distilled water
(5) 30 min Schiff´s reagent (Annex 5)
(6) 2 min 70% ethanol
(7) 2 min 80% ethanol
(8) 2 min 96% ethanol
(9) 2 min isopropanol
(10) Twice 5 min xylene (Riedel-de Häen®)
(11) Mounted with Eukitt (Kindler®)
5.1.3 Pappenheim´s staining (panoptic staining)
This is a differential staining, used for blood and other cytological preparation. It is a
mixture of the Giemsa and May-Grünwald stainings, used to recognize granules in
blood cells, principally in granulocytes. The granules are red, and a good
differentiation of nuclei is possible, the cytoplasm shows different colours according
to the cell type observed (http://flexikon.doccheck.com/Pappenheim-Farbung). The
protocol was as follows:
(1) 3 min May- Grünwald solution
(2) washed with water
(3) 20 min Giemsa solution
67
(4) 2 min 70% ethanol
(5) 2 min 80% ethanol
(6) 2 min 96% ethanol
(7) 2 min isopropanol
(8) Twice 5 min xylene (Riedel-de Häen®)
(9) Mounted with Eukitt (Kindler®)
5.1.4 Immunohistochemistry
Indirect IHC was performed employing a polyclonal rabbit anti-NDV (in house made,
kindly donated by Prof. Dr. Martin Beer, Friedrich-Loeffler-Institut, Federal Research
Institute for Animal Health, Riems, Germany) as primary antibody; the secondary
antibody was a peroxidase labeled polymer-HPR goat anti- rabbit and the reaction
was visualized using DAB (Endvision-Dako®). In order to eliminate background
staining caused by unspecific reactions, the first antibody was pretreated as follows:
(1) Dilution 1:100 in a macerated chorioallantoic membrane (CAM) in PBS
(2) Incubation for 1 hour at room temperature.
(3) Centrifugation at 14 000 RPM (12800 g) in centrifuge was made (Eppendorf
Centrifuge 5410).
(4) The supernatant was collected and diluted again 1:30, in order to obtain a
final concentration of 1:3000 to be used in the IHC technique.
Samples were deparaffinised in xylene and through a series of graded ethanol as
described in the chapter 4.2.1. They were pre-treated against endogenous
peroxidases with 80% ethanol and H2O2 (196 ml ethanol with 4 ml 30% oxygenates
water), subsequently treated as follows:
(1) 3 times washing with PBS, 5 min each time,
(2) incubation in a humid chamber with a solution of chicken serum-PBS (1:5),
68
for 20 min,
(3) incubation for 1 hour with the pre-treated primary antibody
(4) 3 times washing for 5 min with PBS
(5) incubation with the secondary antibody in a humid chamber for 45 min
(6) 3 times washing for 5 min with PBS
(7) incubation with DAB substrate
(8) 1 time washing with PBS, 5 min,
(9) 2 min 70% ethanol
(10) 2 min 80% ethanol
(11) 2 min 96% ethanol
(12) 2 min isopropanol
(13) Placed twice 5 min in xylene (Riedel-de Häen®)
(14) Mounted with Eukitt (Kindler®)
As negative control, tissues from uninfected embryos and fibroblasts with and without
primary antibody were used, and the positive controls were fibroblasts infected with
NDV (as described in the chapter 3).
5.1.5 Lectin histochemistry
The tissues were deparaffinised in xylene and went through via graded ethanol to
water (see chapter 4.2.1). They were pre-treated against endogenous peroxidase
with 80% ethanol and H2O2 (196 ml ethanol with 4 ml 30% oxygenate water). Thus,
the sections were stained as follows:
(1) 3 times 5 min in distilled water
(2) Treatment against endogenous avidin-biotin:
1) 15 min at room temperature with a solution of distilled water (200ml)
and egg white (2 eggs)
2) 3 times 5 min in distilled water
69
3) 15 min at room temperature with a solution of PBS (198 ml) and milk
(1.5% fat, 2 ml) (NOLL and SCHAUB, 2000).
(3) Afterwards they were washed 3 times 5 min in PBS
(4) 1 hour incubation with the lectin (100 μl, 0,1 mg/ml) in a humid chamber at
room temperature The biotinylated lectins used are shown in the table 6.
(5) 3 times 5 min in PBS.
(6) In order to visualize the reactions the samples were:
1) Incubated for 30 min in a humid chamber at room temperature, with one
drop of Biotin Solution (Super Sensitive Link Label IHC Detection
System, BioGenex®)
2) 3 times 5 min in PBS
3) 5 min with Liquid DAB
(7) 5 min in PBS
(8) 10 min in water
(9) 2 min 70% ethanol
(10) 2 min 80% ethanol
(11) 2 min 96% ethanol
(12) 2 min isopropanol
(13) Twice 5 min xylene (Riedel-de Häen®)
(14) Mounted with Eukitt (Kindler®)
Table 6. Lectins used in the study
Lectin Inhibitory carbohydrate Company
Bandeiraea
simplicifolia (BSA-I)
α-D-Galactoside and α-linked galactose
oligosaccharides.
Sigma®
Concanavalia A
(Con A)
α-methyl-mannopyranoside � α-D-Mannose � α-D-
Glucose � α-N-acetyl-D-glucosamine.
Sigma®
Triticum vulgaris
(WGA)
GlcNacβ(1,4) GlcNac � GlcNac �� sialic acid
(Neu5Ac) �� GalNac.
Sigma®
Maackia amurensis
(MAA)
sialic acid α (2,3) galactose EY
laboratories®
70
The negative controls were treated with the same protocol but without lectin
incubation. Instead of lectins, PBS was used. Control inhibition with inhibitory
monosaccharides was made as follows:
(1) All sugars [galactose, mannose, glucose, N-acetyl-glucosamine (glcNAc) and N-
acetyl-galactosamine (galNAc)] were prepared as a 200 mM solution,
(2) Lectins were dissolved at a concentration of 20 μg/ml_1 solution.
(3) The solution of sugars and lectins were mixed in a ratio of 1:1, resulting in a final
concentration of 0.1 mol sugar and 10μg/ml_1 lectin.
(4) The solution was incubated on the vortex for 1 h.
(5) Those pre-incubated lectins were subsequently used in LH as described above.
Removal of neuraminic acid from MAA was made as follows:
(1) Ten units of neuraminidase were dissolve in 70 ml 0,5 M sodium acetate buffer (pH
5,5).
(2) The slides were placed in a humid chamber and incubated overnight at 37°C.
(3) After washing in lectin buffer, the regular MAA staining was carried out as above.
71
5.2 Technovit sections
The samples embedded in Technovit did not require dehydration.
5.2.1 Hematoxylin and Eosin
The principles of the staining method are the same as for the H&E staining of paraffin
sections. The following staining protocol was used:
(1) 60 min hematoxylin (Delafield formula) (Annex 3)
(2) 5-10 sec 0.1% HCL in 70% ethanol
(3) 15 min in water
(4) 5 min in eosin (Annex 4) with 5 drop of acetic acid glacial (Riedel-de
Haën®)
(5) Washed rapidly in tap water
(6) 70% ethanol
(7) 3 min 80% ethanol
(8) Twice 3 min in 96% ethanol
(9) Dry and mounted with DePeX (Serva®)
5.2.2 Toluidine blue
This is a monochromatic stain the reaction of which demonstrates the presence
of proteins. It is possible to observe different tones of blue according to the
isoelectric point of the proteins. The following protocol was used (RICHARDSON et
al., 1960):
72
(1) 2 min in Toluidine blue (Annex 6)
(2) 1 min distilled water
(3) 3 min 70% ethanol
(4) 3 min 80% ethanol
(5) Twice 3 min in 96% ethanol
(6) Dry and mounted with DePeX (Serva®)
6 Data collection and statistical analysis
The histological sections were examined using a light microscope (Zeiss®) in order to
compare the differences between glycosylation patterns, to observe normal histology,
possible histopathology and the presence of glycoconjugates (AB-PAS) in both
groups. This study had a qualitative part and a semi quantitative part. In the former a
description of the histological structure and glycosylation patterns was made, and in
the latter numeric scales were established in order to compare statistically the
differences between both uninfected and infected tissues.
For LH a color scale was established, so that no color was graded as number 0, low
reaction (cream) as 1, middle reaction (light brown) as 2, strong reaction (brown) as 3
and very strong reaction (dark brown) as 4.
Histological observations were also graded in a numerical scale, with lesion-
presence designated as 1 and no lesion-presence as 2. For AB-PAS, according to
the color of the reaction the scale was: 1 for blue (acid), 2 for red (neutral), 3 for violet
(mixed) and 4 in case of no reaction.
73
All data were processed in an Excel® table and analyzed with the SAS-Statistical
Program®. Variance, average and Fischer´s exact test were calculated. For the latter
test a p<0.05 was considered as significant. The statistical analysis was made in
cooperation with the Department of Biometry, Epidemiology and Information
Processing of the University of Veterinary Medicine Hannover Foundation, Germany.
The stained sections were photographed with a digital camera (Olympus® DP70)
adapted to a light microscope (Axioscope Zeiss®).
74
IV. Results
1 Determination of the LD50 of the NDV
1.1 Virus titration in cell culture (chicken fibroblasts)
CPE was observed at 4 days p.i. in the chicken fibroblasts infected with virus
pathotypes Herts 33, Komarov, HB-1 and first passage HB-1, and 6 days p.i. for 1
passage in case of pathotypes Herts 33 and Komarov. Data in detail are shown in
Tables 7, 8 and 9.
LD50 of the NDV in the fibroblast cultures was calculated as described in Materials
and Methods (chapter 2.1). An example of the formula used is shown as follows:
LD50 of the NDV Herts 33 virus pathotype:
PD= [56 - 50%] = 0.1
[56 - 9]
log of 50% endpoint= log 9 - (0.1 x log 1) = -9.1
LD50= 10.1
The LD50 determined for the different pathotypes were NDV Herts 33: 10.1 for the
original NDV stock and, 10.4 for the first passage; NDV Komarov pathotype: 11.9 for
the original NDV stock and, 3.2 for the first passage; NDV HB-1 pathotype: 5 for the
original NDV stock and, 10.1 for the first passage.
75
Tabl
e 7.
Cyt
opat
hic
effe
ct o
bser
ved
in c
hick
en fi
brob
last
cul
ture
s in
fect
ed w
ith N
DV
Her
ts 3
3
Orig
inal
ND
V st
ock
Viru
s fir
st p
assa
ge
[ND
V]
Cel
lsAn
n C
PE
/ to
tal
% C
PE
C
ells
Ann
CP
E/
tota
l% CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
10-1
80
690
69/6
910
0.8
071
071
/71
100
10-2
80
610
61/6
110
08
063
063
/63
100
10-3
80
530
53/5
310
08
055
055
/55
100
10-4
80
450
45/4
510
08
047
047
/47
100
10-5
80
370
37/3
710
08
039
039
/39
100
10-6
80
290
29/2
910
08
031
031
/31
100
10-7
80
210
21/2
110
08
023
023
/23
100
10-8
80
130
13/1
310
08
015
015
/15
100
10-9
44
54
5/9
566
27
27/
978
10-1
01
71
111/
119
17
19
1/10
1010
-11
08
019
0/19
00
80
170/
170
[ND
V]=
ND
V d
ilutio
n, C
PE
= n
umbe
r of w
ells
with
cyt
opat
hic
effe
ct, n
o C
PE
= n
umbe
r of w
ells
with
out c
ytop
athi
c ef
fect
, A
n =
accu
mul
ated
num
ber
of w
ells
from
max
imal
to m
inim
al d
ilutio
n (C
PE
), an
d fro
m m
inim
al to
max
imal
dilu
tion
(no
CP
E) (
VIL
LEG
AS
, 199
8).
76
Tabl
e 8.
Cyt
opat
hic
effe
ct o
bser
ved
in c
hick
en fi
brob
last
cul
ture
s in
fect
ed w
ith N
DV
Kom
arov
.
Orig
inal
ND
V st
ock
Viru
s fir
st p
assa
ge[N
DV
]C
ells
Ann
CP
E/
tota
l%
CP
EC
ells
Ann
CP
E/
tota
l% CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
10-1
80
830
82/8
210
08
015
015
/15
100
10-2
80
750
74/7
410
06
47
47/
1164
10-3
80
670
66/6
610
01
71
111/
128
10-4
80
590
58/5
810
00
80
190/
190
10-5
80
510
50/5
010
00
80
270/
270
10-6
80
430
42/4
210
00
80
350/
350
10-7
80
350
34/3
410
00
80
430/
430
10-8
80
270
26/2
610
00
80
510/
510
10-9
80
190
18/1
810
00
80
590/
590
10-1
07
111
110
/11
910
80
670/
670
10-1
14
44
54/
944
08
075
0/75
0[N
DV
]= N
DV
dilu
tion,
CP
E =
num
ber o
f wel
ls w
ith c
ytop
athi
c ef
fect
, no
CP
E =
num
ber o
f wel
ls w
ithou
t cyt
opat
hic
effe
ct,
An
= ac
cum
ulat
ed n
umbe
r of
wel
ls fr
om m
axim
al to
min
imal
dilu
tion
(CP
E),
and
from
min
imal
to m
axim
al d
ilutio
n (n
o C
PE
) (V
ILLE
GA
S, 1
998)
.
77
Tabl
e 9.
Cyt
opat
hic
effe
ct o
bser
ved
in c
hick
en fi
brob
last
cul
ture
s in
fect
ed w
ith N
DV
HB
-1.
Orig
inal
ND
V st
ock
Viru
s fir
st p
assa
ge[N
DV
]C
ells
Ann
CP
E/
tota
l%
CP
EC
ells
Ann
CP
E/
tota
l% CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
CPE
no CPE
10-1
40
140
14/1
410
06
051
051
/51
100
10-2
40
100
10/1
010
06
045
045
/45
100
10-3
40
60
6/6
100
60
390
39/3
910
010
-42
22
22/
450
60
330
33/3
310
010
-50
40
60/
60
51
271
27/2
896
10-6
04
010
0/10
05
122
222
/24
9210
-70
40
140/
140
51
173
17/1
989
10-8
04
018
0/18
05
112
412
/16
7510
-90
40
220/
220
42
76
7/13
5410
-10
04
026
0/26
03
33
93/
1225
10-1
10
40
300/
300
06
015
0/15
0[N
DV
]= N
DV
dilu
tion,
CP
E =
num
ber o
f wel
ls w
ith c
ytop
athi
c ef
fect
, no
CP
E =
num
ber o
f wel
ls w
ithou
t cyt
opat
hic
effe
ct,
An
= ac
cum
ulat
ed n
umbe
r of
wel
ls fr
om m
axim
al to
min
imal
dilu
tion
(CP
E),
and
from
min
imal
to m
axim
al d
ilutio
n (n
o C
PE
) (V
ILLE
GA
S, 1
998)
.
78
1.2 Inoculation of embryonated chicken eggs
The LD50 for the different pathotypes of NDV in embryonated chicken eggs were
calculated as described before (chapter 1.1), data are showed in details in tables 10,
11 and 12. Calculated LD50 was for NDV Herts 33 pathotype: 12 for the original NDV
stock and, 10 for the first passage; NDV Komarov pathotype: 8.3 for original NDV
stock and, 9.3 for the first passage; NDV HB-1 pathotype: 9.5 for the original NDV
stock and, 10 for the first passage.
Survival time varied between 1.5 and 6 days depending on the pathotype inoculated
and the passage used. In the case of NDV Komarov and HB-1 pathotypes an
increase in the survival time was observed when the first passage of the virus was
used (Fig. 6).
Allantoic fluid from the surviving embryos were tested by HA-test and the percentage
of positivity was over 50% in embryonated eggs inoculated with NDV Komarov
pathotype first passage, NDV HB-1 pathotype original NDV stock and NDV first
passage. In the case of the embryonated eggs inoculated with NDV Herts 33
pathotype (original NDV stock virus and first passage) as well as NDV Komarov
pathotype (original NDV stock) the positive percentage was less than 40% (see Fig.
7).
Table 10. Mortality of chicken embryos in eggs inoculated with NDV Herts 33 Original NDV stock Virus first passage
[NDV] Embryos An Embryos Acc. n nd nl nd nl nd/total % died nd nl nd nl nd/total % died
10 -6 - - - - - - 4 0 15 0 15/15 10010 -7 3 1 17 1 17/18 94 4 0 11 0 11/11 10010 -8 3 1 14 2 14/16 88 3 1 7 1 7/8 8810 -9 3 1 11 3 11/14 79 1 3 4 4 4/8 5010 -10 4 0 8 3 8/11 73 3 1 3 5 3/8 3810 -11 3 1 4 4 4/8 50 0 4 0 9 0/9 010 -12 1 4 1 8 1/8 13 - - - - - -
[NDV]= NDV dilution, nd= number of dead embryos, nl= number of live embryos, An = Accumulated number of embryos from maximal to minimal dilution (nd), and from minimal to maximal dilution (nl) (VILLEGAS, 1998).
79
Table 11. Mortality of chicken embryos in eggs inoculated with NDV Komarov Original NDV stock Virus first passage
Embryos An Embryos An[NDV] nd nl nd nl nd/total % died nd nl nd nl Nd/total % died 10 -4 - - - - - - 3 1 18 1 18/19 9510 -5 - - - - - - 2 2 15 3 15/18 8310 -6 4 0 7 0 7/7 100 3 1 13 4 13/17 7610 -7 2 2 3 2 3/5 60 3 1 10 5 10/15 6710 -8 1 3 1 5 1/5 20 4 0 7 5 7/12 5810 -9 0 4 0 9 0/9 0 3 1 3 6 3/9 3310 -10 0 4 0 13 0/13 0 - - - - - -10 -11 0 4 0 17 0/17 0 - - - - - -
[NDV]= NDV dilution, nd= number of dead embryos, nl= number of live embryos, An= Accumulated number of embryos from maximal to minimal dilution (nd), and from minimal to maximal dilution (nl) (VILLEGAS, 1998).
Table 12. Mortality of chicken embryos in eggs inoculated with NDV HB-1 Original NDV stock Virus first passage
Embryos An Embryos An[NDV] nd nl nd nl nd/total % died nd nl nd nl Nd/total % died 10 -5 4 0 15 0 15/15 100 - - - - - -10 -6 3 1 11 1 11/12 92 4 0 15 0 15/15 10010 -7 2 2 8 3 8/11 73 2 2 11 2 11/13 8510 -8 2 2 6 5 6/11 55 4 0 9 2 9/11 8210 -9 4 0 4 5 4/9 44 1 3 5 5 5/10 5010 -10 - - - - - - 4 0 4 5 4/9 4410 -11 - - - - - - 0 4 0 9 0/9 0
[NDV]= NDV dilution, nd= number of dead embryos, nl= number of live embryos, An= Accumulated number of embryos from maximal to minimal dilution (nd), and from minimal to maximal dilution (nl) (VILLEGAS, 1998).
80
Herts 33/originalstock
Herts 33/virus first passage
Komarov/originalstock
Komarov/virus first passage
HB-1/original stock
HB-1/virus first passage
0
1
2
3
4
5
6
7
Chi
cken
em
bryo
sur
viva
l tim
e (d
ays)
Figure 6. Survival time of chicken embryos infected with NDV at day 11th of incubation
Herts 33/ original stock
Herts 33/virus first passage
Komarov/ originalstock
Komarov/virus first passage
HB-1/original stock HB-1/
virus first passage
0
10
20
30
40
50
60
70
% o
f pos
itiv
HA
-test
Figure 7. Result of HA-test of chicken embryos infected with NDV at day 11th
of incubation
81
2 Cytological study
Specific reactions were observed in the fibroblasts infected with the different
pathotypes of the NDV, while no reaction was detected in the uninfected fibroblasts,
indicating the specificity of the antibody. Fibroblasts infected with NDV HB-1
pathotype showed a weaker reaction than those infected with NDV Komarov
pathotype (Fig. 8).
a b c
Figure 8. Immunocytochemistry of chicken fibroblasts (Magnification 400X)a. Control stained with Hematoxilin, b. Positive fibroblasts infected with pathotype HB-1, c. Positive fibroblasts infected with pathotype Komarov.
82
3 Studies of chicken embryos
3.1 Macroscopic observations
Hemorrhagic lesions were observed in the skin and the egg membrane of 10
embryos infected at day 11th with Komarov virus, and 3 embryos infected with HB-1
virus. All other infected embryos and the controls did not show lesions (Fig. 9).
Figure 9. Hemorrhagic lesions in chicken embryos infected with NDV.
Arrows point at hemorrhagic lesions present in neck, near to eye and eggs membrane are present.
3.2 Results of the HA-Test
The presence of NDV in allantoic fluid was determined with the HA-Test in 17 NDV
Komarov pathotype inoculated embryonated eggs at 11th i.d. and 10 inoculated at
15th i.d.; 15 NDV HB-1 pathotype inoculated eggs at 11th i.d. and 11 inoculated at 15th
83
i.d. All controls were negative. Two embryos per group were embedded in Technovit
and the other ones were embedded in paraffin wax for histological examination.
3.3 Immuno histochemical results
From the HA-Test positive eggs infected at 11th i.d. with NDV Komarov pathotype
collected at 15th i.d. more than 50% of the evaluated esophagi, proventriculi,
tracheae, lungs and air sacs were positive for viral antigen. From the embryos
infected with NDV HB-1 pathotype collected at day 15th i.d. more than 50% the
esophagi, tracheae and air sacs were positive for NDV by IHC (Table 13). The
positive reaction, in all positive tissues, was only observed in the epithelial cells (Fig.
10). Duodenum and cecum showed unspecific reactions, positive reactions were
observed also in the control group. Therefore, it was not possible to distinguish
between positive and unspecific reaction in these cases. All other negative controls
were negative for NDV.
Table 13. Number of positive tissues infected with the different pathotype of NDV by IHC.
Number of positive tissue infected with NDV Komarov
pathotype
Number of positive tissue infected with NDV HB-1
pathotype Observation
Organ Day15(n=15)
19(n=8)
15(n=13)
19(n=9)
Esophagus 12 0 10 1Ventriculus 7 0 6 1Proventriculus 8 0 6 0Duodenum* 3 0 1 0Cecum* 7 3 9 5Liver 6 0 3 0Trachea 15 1 10 1Lung 12 0 5 2Air Sac 12 0 7 0Thymus 2 0 0 0Bursa of Fabricius 1 0 0 0Kidney 0 0 0 0Spleen 2 0 2 0 * unspecific reaction.
84
Figure 10. Results of IHC in chicken infected embryos a. Positive reaction (see arrow) in epithelium of the esophagus infected with NDV Komarov pathotype, b. Positive reaction (see arrow) in epithelium of the esophagus infected with NDV HB-1 pathotype, c. Negative reaction (see arrow) in epithelium of the esophagus in the control group, d. Positive reaction (see arrow) in epithelium of the trachea infected with NDV Komarov pathotype, e. Positive reaction (see arrow) in epithelium of the trachea infected with NDV HB-1 pathotype, f. Negative reaction (see arrow) in epithelium of the trachea in the control group.
85
3.4 Histological and histochemical observations
3.4.1 Respiratory system
3.4.1.1 Histological observations
At 11th i.d., the control embryos showed an open tracheal lumen and the cells formed
a meshwork, the epithelium was pseudostratified with cuboidal cells and had few
layers. Goblet cells were not present and the cartilaginous ring had not developed.
By the thirteenth incubation day the cartilaginous ring was more compact, the
epithelium was pseudostratified and columnar, but goblet cells could not be detected
until 15th i.d. Liquid in the tracheal lumen was observable beginning at the 15th i.d., as
well as erythrocytes and granulocytes (Fig. 11).
Changes in histological structure were detected in 5/15 tracheas of embryos infected
with NDV Komarov pathotype and 4/13 tracheas of embryos infected with NDV HB-1
pathotype at 15th i.d. The alterations observed were the absence of a ciliated
epithelium and goblet cells. The epithelium was cubic.
The structure of the complete development lungs was recognized at 19th i.d.,
nevertheless, the presence of parabronchi was visible since 11th i.d. They showed a
simple cuboidal epithelium. The air capillaries and the presence of atria were
observed from 11th i.d. The air capillaries showed a simple squamous epithelium (Fig.
12). The air sacs were seen from 11th i.d., they show a cuboidal or simple squamous
epithelium depending on the region observed. Lesions were not found in the infected
embryos. Erythrocytes and granulocytes were seen in lungs and air sacs from 15th
i.d. on the control and infected group.
86
Figu
re 1
1. T
rach
ea d
evel
opm
ent o
f uni
nfec
ted
SPF
chic
ken
embr
yos
a. E
mbr
yo 1
1th i.
d., H
&E
; b. E
mbr
yo 1
3th i.
d., H
&E
; c. E
mbr
yo 1
5th i.
d., H
&E
; d.
Em
bryo
17th
i.d.
, H&
E; e
. Em
bryo
11th
i.d.
,H&
E;
f. E
mbr
yo 1
5th i.
d., W
GA
. 1-
Epi
thel
ium
, 2-G
oble
t cel
ls, 3
-Tel
a su
bmuc
osa,
4-C
artil
agin
ous
ring,
E
ryth
rocy
tes.
87
Figure 12. Development of lung parabronchial tissue in uninfected SPF embryos. H&E. a. Embryo 11th i.d., b. Embryo 15th i.d., c. Embryo 17th i.d., d. Embryo 11th i.d. Erythrocytes, Granulocytes.
3.4.1.2. Lectin histochemical observations
Con A (α-methyl-mannopyranoside and α-D-Mannose binding) labeled strongly or
very strongly in the control group: a) the apical part of the epithelial cells of
parabronchi (lung) and air sacs from embryos, independent of the incubation day;
b) the apical part of the tracheal epithelial cells of embryos at 11th, 13th, 15th and 19th
i.d.; c) the cytoplasm of air sac epithelial cells of embryos at 13th and 15th i.d.; and d)
the tunica muscularis of the trachea at 11th i.d. All other structures in the trachea, lung
88
and air sacs were not labeled or reacted only weak, low or medium intensities (Table
14).
BSA-I (α-D-galactoside and α-linked galactose binding) labeled strongly or very
strongly: a) the apical part of the tracheal epithelial cells of embryos at 11th, 13th, 17th
and 19th i.d.; b) the tracheal goblet cells of embryos at 19th i.d.; c) the tunica
muscularis in trachea of embryos at 11th i.d.; d) the apical part of the epithelial cells of
parabronchies (lung) of embryos at 11th, 15th, 17th and 19th i.d.; e) the apical part of
air sac epithelial cells of embryos at 11th, 13th, 15th and 19th i.d.; and f) the cytoplasm
of air sac epithelial cells of embryos at 11th and 13th i.d. (Table 14).
WGA (GlucNac binding) labeled strongly or very strongly: a) the apical part of the
epithelial cells of trachea, parabronchi (lung) and air sac of embryos, independent of
the incubation day; b) the tracheal goblet cells of embryos at 15th and 17th i.d.; and
c) the cytoplasm of cells in parabronchi and air sacs at 17th and 13th i.d., respectively
(Table 14).
MAA (sialic acid α (2,3) galactose binding) labeled strongly or very strongly: a) the
apical part of the epithelial cells of parabronchies (lung) of embryos at all incubation
day; and b) the air sac at 13th i.d. All other structures in the trachea, lung and air sacs
were not labeled or reacted only weak, low or medium intensities (Table 14).
In the control groups, significant differences (Fisher Exact Test�0.05) for the staining
with BSA-I were observed in the tela submucosa of the trachea, in the tracheal
cartilaginous ring, in the apical part of the lung epithelial cells between different
incubation days. Furthermore, in the same group significant differences (Fisher Exact
Test�0.05) for the staining with WGA were observed in the apical part of the air sac
epithelial cells between 11th and 13th i.d. But differences were also found for MAA in
the apical part and cytoplasm of the tracheal epithelial cells, in the cytoplasm of the
lung epithelial cells and in the apical part of the air sac epithelial cells when
comparing the different incubations days (Table 14).
89
Comparing the lectin staining pattern of control groups and the infected (at day 11th
of incubation with NDV Komarov and HB-1 pathotypes), significant differences
(Fisher Exact Test�0.05) were observed in: a) the apical part of the tracheal
epithelial cells stained with Con A and BSA-I in the NDV Komarov pathotype infected
group and with Con A in the NDV HB-1 pathotype infected group, b) tela submucosa
in trachea stained with Con A in the NDV Komarov pathotype infected group, c)
tracheal cartilaginous ring stained with WGA in the NDV Komarov pathotype infected
group, d) tracheal tela muscularis stained with BSA-I in the NDV Komarov infected
group, e) cytoplasm of the epithelial cells in the lung parabronchies stained with MAA
in the NDV HB-1 pathotype infected group and f) the apical part and cytoplasm of the
epithelial cells in air sac stained with WGA in the NDV HB-1 pathotype infected
group. Details of the data are summarized in Table 15. The number of positive
tracheae examined in IHC of the embryos infected at 15th i.d. was not sufficient for
the statistic analysis.
90
Tabl
e 14
. Lec
tin h
isto
chem
ical
resu
lts o
f the
resp
irato
ry s
yste
m o
f uni
nfec
ted
chic
ken
embr
yos
of d
iffer
ent d
ays
of in
cuba
tion
Lect
inC
onA
BS
A-1
WG
AM
AA
Day
of i
ncub
atio
n O
r-ga
nS
truct
ure
1113
1517
1911
1315
1719
1113
1517
1911
1315
1719
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=13) sd
=1sd
=1S
d=1
3), 4
)
sd=1
3), 4
)
sd=1
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd
=1sd
=1sd
=1sd
=1S
d=1
4) sd=1
gobl
et c
ells
*
**
*sd
=1sd
=1*
*S
d=1
**
sd=1
tela
sub
muc
osa
sd=1
sd=1
sd=1
sd=1
2),
3),
5)sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1S
d=1
sd=1
sd=1
sd=1
sd=1
sd=1
carti
lagi
nous
rin
gsd
=1sd
=1sd
=1sd
=15) sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1S
d=1
sd=1
sd=1
T R A C H E A
tuni
cam
uscu
laris
sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1ap
ical
par
t of t
he
epith
elia
l cel
ls
(par
abro
nchi
)sd
=1sd
=14) sd
=14) sd
=15)
sd=1
sd=1
sd=1
Sd=
1sd
=1sd
=1sd
=1L U N G
cyto
plas
m o
f the
ep
ithel
ial c
ells
(p
arab
ronc
hi)
sd=1
sd=1
3), 4
)
sd=1
sd=1
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=12) sd
=1sd
=15)
5) sd=1
5)
sd=2
A I R S A C
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
n
o st
aini
ng (a
vera
ge 0
)
low
sta
inin
g (a
vera
ge 1
)
med
ium
sta
inin
g (a
vera
ge 2
)
stro
ng s
tain
ing
(ave
rage
3)
v
ery
stro
ng s
tain
ing
(ave
rage
4).
sd
= st
anda
rd d
evia
tion
with
in t
he s
ame
grou
p, *
= n
o da
ta,
1)=
sign
ifica
nt d
iffer
ence
with
11th
i.d.
, 2)
= s
igni
fican
t di
ffere
nce
with
13th
i.d.
, 3)
= s
igni
fican
t di
ffere
nce
with
15th
i.d.
, 4) =
sig
nific
ant d
iffer
ence
with
17th
i.d.
, 5) =
sig
nific
ant d
iffer
ence
with
19th
i.d.
91
Tabl
e 15
. Lec
tin h
isto
chem
ical
resu
lts o
f the
resp
irato
ry s
yste
m o
f inf
ecte
d ch
icke
n em
bryo
s of
diff
eren
t day
s of
incu
batio
n P
atho
typ
Kom
arov
H
B-1
Lect
inC
on A
B
SA
-1W
GA
MA
AC
on A
B
SA
-1W
GA
MA
AO
rgan
Stru
ctur
eIn
cuba
tion
days
In
cuba
tion
days
15
1915
1915
1915
1915
1915
1915
1915
19ap
ical
par
t of t
he
epith
elia
l cel
ls
1) sd=1
**1) sd
=1**
****
1) sd=1
**sd
=1**
sd=1
**sd
=1**
cyto
plas
m o
f the
ep
ithel
ial c
ells
**
**sd
=1**
**sd
=1**
sd=1
**sd
=1**
sd=1
**go
blet
cel
ls
sd=1
****
****
****
****
tela
sub
muc
osa
1)
****
sd=1
**sd
=1**
****
sd=1
**sd
=1**
carti
lagi
nous
ring
sd
=1**
**1)
****
sd=1
****
****
T R A C H E A
tuni
ca m
uscu
laris
**
1)
****
sd=1
****
****
sd=1
**ap
ical
par
t of t
he
epith
elia
l cel
ls
(par
abro
nchi
)sd
=1*
sd=1
*sd
=1*
sd=1
*sd
=1sd
=1sd
=1sd
=1sd
=1L U N G
cyto
plas
m o
f the
ep
ithel
ial c
ells
(p
arab
ronc
hi)
**
sd=1
*sd
=1*
sd=1
sd=1
1) sd=1
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1*
sd=1
*sd
=1*
sd=1
*sd
=1*
sd=1
*
1) sd=1
*sd
=1*
A I R S A C
cyto
plas
m o
f the
ep
ithel
ial c
ells
*
**
sd=1
**
sd=1
*
1) sd=1
*sd
=1*
n
o st
aini
ng (a
vera
ge 0
)
low
sta
inin
g (a
vera
ge 1
)
m
ediu
m s
tain
ing
(ave
rage
2)
s
trong
sta
inin
g (a
vera
ge 3
)
ver
y st
rong
sta
inin
g (a
vera
ge 4
).
sd=
stan
dard
dev
iatio
n w
ithin
the
sam
e gr
oup,
*=
no d
ata,
**
= no
t suf
ficie
nt n
umbe
rs o
f ob
serv
atio
ns to
cal
cula
ted
sd ,
1)=
sign
ifica
nt
diffe
renc
e w
ith th
e co
ntro
l gro
up.
92
3.4.2 Digestive system
3.4.2.1. Histological observations
The control chicken embryos showed luminated esophagi at 11th i.d. At this time, the
stratified epithelium had only a few layers, the number of layers increased during
development. The mucous glands were present for the first time at 15th i.d. At day
19th of incubation, the gland buds exhibited secondary projections and the lumina
began to develop and were formed of cylindrical cells containing granules of
mucinogen (Fig. 13). The lamina muscularis was visible first at 13th i.d. and the tunica
muscularis at 11th i.d. was observed as independent myoblasts. Tunica serosa was
present.
In the proventriculus simple columnar epithelium was seen at 11th i.d.; proventricular
glands were already present extending throughout the lamina propria during
development. At 15th i.d., an increase in number was observed with a stronger
complexity of the glands until 19th i.d. The lamina propria, tunica submucosa, tunica
muscularis and tunica serosa showing the typical structure of the develop organ at
11th i.d.
93
Figure 13. Esophagus development of uninfected SPF chicken embryos a. Embryo 11th i.d., H&E; b. Embryo 13th i.d., H&E; c. Embryo 19th i.d., H&E; d. Embryo 17th i.d., AB-PAS. 1-Epithelium, 2- Lamina muscularis mucosae, 3-Tela submucosa, 4-Tunica muscularis extern, 5-Tunica serosa, Erythrocytes in blood venes.
In the ventriculus from embryos at 11th i.d., the ventricular musculature had already
developed, and during the following time, an increase in thickness was also
observed. The epithelium at 11th i.d. was thin and the layers present increased in
thickness with the development. The glandular cells were observed for the first time
at day 13th of incubation, showing already mucin production. They progressively
projected into the gizzard lumen during development and formed strands of cuboidal
epithelium; a production of keratinoid substance started at day 19th of incubation. The
94
lamina propria was a thin dense layer, and the tela submucosa was not clearly visible
(Fig. 14).
a b
Figure 14. Ventriculus development of uninfected SPF chicken embryos. AB-PASa. Embryo 13th i.d., b. Embryo 19th i.d. Goblet cells.
In order to evaluate always a similar segment of the small intestine (duodenum), the
pancreas was used as a structure of reference. For the large intestine, the reference
used was the ventro-lateral part of the ventriculus (cecum). The epithelial cells
observed from 11th i.d. in both segments were simple cuboidal. At day 13th i.d., the
intestinal villi were present in the duodenum. At day 11th i.d. folds were observed in
the cecum, which grew up rapidly and at 15th i.d. short intestinal villi were present.
Crypts of Lieberkühn were not found at all in the duodenum, but at day 17th of
incubation, immature goblet cells could be recognized. In the cecum crypts of
Lieberkühn and immature goblet cells were observed from 13th and at 15th i.d., they
were positive to AB-PAS. Goblet cells were observed in duodenum at 19th i.d. All
others structures were recognized from 11th i.d. Erythrocytes and granulocytes were
seen from 15th i.d. (Fig. 15).
95
At 11th i.d., hepatocytes were dispersed throughout the hepatic parenchyma, showing
cytoplasmic vesicles. From day 15th i.d., the hepatocytes appeared more organized
and the cytoplasm was more condensed.
Histopathological changes were not observed in any part of the digestive system in
the infected group. Erythrocytes and granulocytes were present in the infected and
control embryos.
Figure 15. Large intestine development of uninfected SPF chicken embryos. H&Ea. Embryo 13th i.d., b. Embryo 19th i.d.1-Enterocytes, 2- Crypts of Lieberkühn.
3.4.2.2. Lectin histochemical observations
Con A (α-D-mannose and α-D-glucose binding) labeled strongly or very strongly:
a) the apical part of the epithelial cells of the esophagus of embryos at all i.d.
evaluated; of the ventriculus of embryos at 13th i.d., the proventriculus at 11th, 13th
and 19th i.d., the small intestine at 11th, 13th and 17th i.d., the large intestine at 11th,
13th and 19th i.d.; b) the tela submucosa of the esophagus of embryos at 11th, 13th
and 19th i.d., the ventriculus at 11th i.d.; c) the goblet cells of the proventriculus of
96
embryos at 11th, 13th, 17th and 19th i.d.; d) the immature goblet cells in the small
intestine at 13th i.d. and the large intestine at 13th and 19 i.d., and e) the tunica
serosa of the esophagus, small and large intestine of embryos at all i.d. evaluated
(Table 16).
BSA-I (α-D-galactoside binding) labeled strongly or very strongly: a) the apical part
of the epithelial cells of the esophagus at 11th i.d., the proventriculus at 11th and
19th i.d., the small intestine at 11th and 17th i.d., and the large intestine at 11th and
13th i.d., b) the tela submucosa of the esophagus at 19th i.d., the ventriculus at
11th i.d., c) the gland cells of the ventriculus at 11th i.d., the proventriculus at 11th and
19th i.d., the small intestine 17th i.d., and d) the tunica serosa of the esophagus and
the small and large intestine at all i.d. evaluated (Table 16).
WGA (GlucNac binding) labeled strongly or very strongly: a) the apical part of the
epithelial cells of the trachea at all i.d. evaluated, the ventriculus at 13th and 17th i.d.,
the proventriculus 13th i.d., the small intestine at 11th and 13th i.d., and the large
intestine at 11th, 13th and 19th i.d.; b) the tela submucosa of the esophagus at 19th i.d.,
the large intestine at 11th i.d.; c) the gland cells of the ventriculus at 13th and 15th i.d.,
the proventriculus at 15th i.d., the small intestine at 11th, 13th and 15th i.d. and the
large intestine at 13th, 15th and 19th i.d.; and d) the tunica serosa of the esophagus,
the small and the large intestine at all i.d. evaluated (Table 16). Goblet cells were no
detectable with AB-staining before the 17th i.d., but were observed with WGA staining
(Fig. 16).
MAA (sialic acid α (2,3) galactose binding) labeled strongly or very strongly: a) the
apical part of the epithelial cells of the ventriculus at 13th, 15th, 17th and 19th i.d., and
b) the gland cells of the ventriculus at all i.d. evaluated, and the proventriculus at 19th
i.d. (Table 16).
In the control group, significant differences (Fisher Exact Test�0.05) for the staining
with Con A were observed for the apical part and cytoplasm of the epithelial cells of
97
the proventriculus and the large intestine, the goblet cells and the tela submucosa
between different i.d. Furthermore, in the same group, significant differences (Fisher
Exact Test�0.05) for the staining with BSA-I were found for the apical part of the
epithelial cells of the ventriculus, the goblet cells of the ventriculus and the
proventriculus when compared at different i.d. For WGA stains, significant differences
were found for the apical part of the epithelial cells of the esophagus, the ventriculus
and the large intestine, the goblet cells of the proventriculus and the large intestine,
the muscularis externa in the large intestine compares at different i.d. (Table 16).
Additionally, differences were observed for MAA stains in the apical part and
cytoplasm of the epithelial cells of the ventriculus, the proventriculus, the lamina
muscularis mucosae of the esophagus and tunica serosa of the small intestine when
compared at different i.d. (Table 16).
In the infected group, no data were collected for embryos infected at 15th i.d., since all
organs of the embryos of these groups (infected with NDV Komarov and HB-1
pathotypes) were negative for NDV by IHC. Both infected groups (infected at day 11th
of incubation) differed significantly (Fisher Exact Test�0.05) from the control group
when the staining with different lectins was compared. In the apical part of the
epithelial cells and the goblet cells of the ventriculus a stronger MAA staining was
seen for both infected groups and stronger staining where observed with BSA-I in the
ventricular goblet cells in the group infected with NDV Komarov pathotype (Details in
Table 17).
98
1
1
2
33 2
1
1
3
Figure 16. Large intestine, reaction of goblet cells in uninfected SPF chicken embryosa. Large intestine from chicken embryo at 13th i.d. AB-PAS staining, b. Large intestine from chicken embryo at 13th i.d. WGA staining, c. Large intestine from chicken embryo at 19th i.d. AB-PAS staining, d. Large intestine from chicken embryo at 19th
i.d. WGA staining.Structure observed: 1. Enterocytes, 2. Goblet cells, 3. Tela submucosa
99
Tabl
e 16
. Lec
tin h
isto
chem
ical
resu
lts o
f the
dig
estiv
e sy
stem
of u
ninf
ecte
d ch
icke
n em
bryo
s of
diff
eren
t day
s of
incu
batio
n Le
ctin
Con
AB
SA
-1W
GA
MA
AIn
cuba
tion
days
O
r-ga
nS
truct
ure
1113
1517
1911
1315
1719
1113
1517
1911
1315
1719
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1sd
=1sd
=1sd
=15)
5)
sd=1
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd=1
sd=1
Lam
ina
mus
cula
ris
muc
osae
sd=1
sd=1
sd=1
4)
sd=1
tela
sub
muc
osa
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
tuni
ca m
uscu
laris
ex
tern
asd
=1sd
=1sd
=1
E S O P H A G U Stu
nica
ser
osa
sd=1
sd=1
sd=1
sd=1
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1sd
=1sd
=1sd
=1
2) sd=1
sd=1
sd=1
sd=1
2)
sd=1
sd=1
sd=1
sd=1
3), 4
),
5) sd=1
sd=1
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd
=1sd
=1sd
=1sd
=1sd
=15)
gobl
et c
ells
3) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=0
tela
sub
muc
osa
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
V E N T R I C U L U Stu
nica
mus
cula
ris
exte
rna
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
100
Con
tinua
tion…
Tab
le 1
6. L
ectin
his
toch
emic
al re
sults
of t
he d
iges
tive
syst
em o
f uni
nfec
ted
chic
ken
embr
yos
of d
iffer
ent d
ays
of
i
ncub
atio
n Le
ctin
Con
A
BS
A-1
WG
AM
AA
1113
1517
1911
1315
1719
1113
1517
1911
1315
1719
Or-
gan
Stru
ctur
e In
cuba
tion
days
ap
ical
par
t of t
he
epith
elia
l cel
ls
2), 3
)
sd=1
sd=1
sd=1
5) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
3) sd=1
5) sd=1
sd=1
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
P R O V E N T R I C U L U S
gobl
et c
ells
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
4) sd=1
sd=1
5)
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
apic
al p
art o
f the
ep
ithel
ial c
ells
3)
, 4),
5)
3) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd
=1cy
topl
asm
of t
he
epith
elia
l cel
ls5)
5)5)
sd=1
sd=1
sd=1
sd=1
sd=1
sd=0
gobl
et c
ells
(im
mat
ure)
**sd
=14) sd
=1**
**sd
=1sd
=1**
**sd
=1**
****
Lam
ina
mus
cula
ris
muc
osae
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
Tela
sub
muc
osa
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
Tuni
cam
uscu
laris
ex
tern
asd
=1sd
=1sd
=1sd
=1
S M A L L I N T E S T I N Etu
nica
ser
osa
sd=1
sd=1
sd=1
sd=1
3) sd=1
sd=1
4)
sd=1
sd=1
101
Con
tinua
tion…
.Tab
le 1
6. L
ectin
his
toch
emic
al re
sults
of t
he d
iges
tive
syst
em o
f uni
nfec
ted
chic
ken
embr
yos
of d
iffer
ent d
ays
of
in
cuba
tion
Lect
inC
on A
B
SA
-1W
GA
MA
A11
1315
1719
1113
1517
1911
1315
1719
1113
1517
19O
r-ga
n S
truct
ure
Incu
batio
n da
ys
apic
al p
art o
f th
e ep
ithel
ial
cells
4) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
4)
sd=1
5) sd=1
2), 3
)
sd=1
sd
=1cy
topl
asm
of
the
epith
elia
l ce
llssd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1
gobl
et c
ells
**
sd=1
sd=1
**sd
=1sd
=1**
sd=1
sd=1
5) sd=2
sd=1
**sd
=1im
mat
ure
gobl
et c
ells
**
sd=1
sd=1
sd=1
**sd
=1**
sd=1
**la
min
am
uscu
laris
m
ucos
aesd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1te
lasu
bmuc
osa
4) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
tuni
cam
uscu
laris
ex
tern
asd
=1sd
=1sd
=1sd
=14)
sd=1
sd=1
sd=1
sd=1
sd=1
L A R G E I N T E S T E S T I N Etu
nica
ser
osa
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
L I V E R
Hep
atoc
ytes
no
stai
ning
(ave
rage
0)
lo
w s
tain
ing
(ave
rage
1)
m
ediu
m s
tain
ing
(ave
rage
2)
s
trong
sta
inin
g (a
vera
ge 3
)
ver
y st
rong
sta
inin
g (a
vera
ge 4
).
sd=
stan
dard
dev
iatio
n w
ithin
the
sam
e gr
oup,
*=
no d
ata,
1)=
sign
ifica
nt d
iffer
ence
with
11th
i.d.
, 2) =
sig
nific
ant d
iffer
ence
with
13th
i.d.
, 3) =
sig
nific
ant
diffe
renc
e w
ith 1
5th i.
d.,
4) =
sig
nific
ant d
iffer
ence
with
17th
i.d.
, 5) =
sig
nific
ant d
iffer
ence
with
19th
i.d.
102
Tabl
e 17
. Lec
tin h
isto
chem
ical
resu
lts o
f the
dig
estiv
e sy
stem
of i
nfec
ted
chic
ken
embr
yos
of d
iffer
ent
da
ys o
f inc
ubat
ion
Pat
hoty
pK
omar
ov
HB
-1Le
ctin
Con
A
BS
A-1
WG
AM
AA
Con
A
BS
A-1
WG
AM
AA
Incu
batio
n da
ys
Incu
batio
n da
ys
Org
anS
truct
ure
1519
1519
1519
1519
1519
1519
1519
1519
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1*
sd=1
*sd
=1*
*sd
=1**
sd=1
**sd
=1**
sd=1
**
cyto
plas
m o
f the
ep
ithel
ial c
ells
**
**
****
sd=1
**sd
=1**
lam
ina
mus
cula
ris
muc
osae
**
sd=1
*sd
=1*
****
sd=1
**sd
=1**
tela
sub
muc
osa
sd=1
*sd
=1*
sd=1
*sd
=1*
sd=1
**sd
=1**
**sd
=1**
tuni
ca m
uscu
laris
ex
tern
a*
*sd
=1*
sd=1
***
****
sd=1
**
E S O P H A G U Stu
nica
ser
osa
sd=1
*sd
=1*
*sd
=1*
****
**sd
=1**
apic
al p
art o
f the
ep
ithel
ial c
ells
sd
=1
**
sd=1
*1) sd
=1
***
sd=1
**
sd=1
**1) sd
=1
**
cyto
plas
m o
f the
ep
ithel
ial c
ells
sd
=1
*
sd=1
**
sd=1
***
**
sd=1
**
sd=1
**
gobl
et c
ells
sd
=1*
1) sd=1
*sd
=1*
1) sd=1
***
**sd
=1**
sd=1
**
tela
sub
muc
osa
sd=1
*sd
=1*
**
sd=1
**sd
=1**
sd=1
****
V E N T R I C U L U S
tuni
ca m
uscu
laris
ex
tern
a*
**
***
****
**
103
Con
tinua
tion
Tabl
e 17
. Lec
tin h
isto
chem
ical
resu
lts o
f the
dig
estiv
e sy
stem
of i
nfec
ted
chic
ken
embr
yos
o
f diff
eren
t day
s of
incu
batio
n P
atho
typ
Kom
arov
H
B-1
Lect
inC
on A
B
SA
-1W
GA
MA
AC
on A
B
SA
-1W
GA
MA
AIn
cuba
tion
days
In
cuba
tion
days
O
rgan
Stru
ctur
e15
1915
1915
1915
1915
1915
1915
1915
19ap
ical
par
t of
the
epith
elia
l ce
lls
sd=1
*sd
=1*
sd=1
*sd
=1*
sd=1
*sd
=1*
sd=1
*sd
=1*
cyto
plas
m o
f th
e ep
ithel
ial
cells
sd=1
**
*sd
=1*
sd=1
**
*sd
=1*
P R O V E N T R I C U L U Sgo
blet
cel
lssd
=1*
sd=1
*sd
=1*
sd=1
*sd
=1*
sd=1
**
sd=1
*L I V E R
Hep
atoc
ytes
sd=1
**
**
***
**
*
no s
tain
ing
(ave
rage
0)
low
sta
inin
g (a
vera
ge 1
)
med
ium
sta
inin
g (a
vera
ge 2
)
stro
ng s
tain
ing
(ave
rage
3)
ve
ry s
trong
sta
inin
g (a
vera
ge 4
).
sd=
stan
dard
dev
iatio
n w
ithin
the
sam
e gr
oup,
*=
no d
ata,
**
= no
t su
ffici
ent
num
bers
of
obs
erva
tions
to
calc
ulat
ed s
d ,
1)=
sign
ifica
nt d
iffer
ence
with
the
cont
rol g
roup
.
104
3.4.3 Immune system
3.4.3.1 Histological observations
In the control group, lymphocytes were observed in the thymus at 11th i.d., but a clear
differentiation of the thymic lobes was not noticeable. The lymphocytes observed
were of both types, large and small. At 13th i.d., the lobes were more defined but the
cortical and medullar zones were undistinguishable. From day 15th of incubation, a
differentiation into cortex and medulla became evident.
The spleen of the chicken embryos of the control group appeared as a round organ
from 11th i.d., reticular cells and erythrocytes were present, as well as arterial vessels
and veins, but a clear differentiation of the red and white pulp was not observed.
Regarding the bursa of Fabricius of the control group, a capsule was observed at day
11th of incubation, showing a cuboidal epithelium and connective tissue. At 13th i.d.,
the Tunica serosa, the Tunica muscularis and the first bursal follicles were observed.
High numbers of granulocytes were present from 13th i.d. (Fig. 17).
These organs of infected chicken did not show any histopathological alterations in
comparison to non-infected ones. Erythrocytes and granulocytes were observed in
both the control and the infected group.
3.4.3.2. Lectin histochemical observations
Thymus and spleen presented a low or medium staining with all the lectins used in
this study. The apical part of the epithelium of the bursa of Fabricius presented a
strong or very strong labeling with all lectins used at the different observations days
(see Table 18). In the infected group, no differences were observed, but the number
of observations was small because of the low number of organs IHC positive.
105
Figure 17. Bursa of Fabricius of uninfected SPF chicken embryos a. Embryo 13th i.d., H&E; b. Embryo 19th i.d., H&E; c. Embryo 19th i.d., Pappenheim staining1-Epithelium, 2-Follicles, 3- Granulocytes
3.4.4 Urinary system (Kidney)
3.4.4.1 Histological observations
In the control group from 11th i.d. on, the morphology of the kidney was well defined;
exhibiting well differentiated structures specific for the glomeruli (small and large), the
proximal and distal tubules showing the typically morphology as know from chicken
adult. From 15th i.d. the presence of erythrocytes and granulocytes was noticeable.
No histopathological alterations were observed in the infected group.
3.4.4.2 Lectin histochemical observations
A strong or very strong staining was observed in: a) the apical part of the epithelium
of the proximal tubules with WGA at 11th i.d.; b) the apical part and cytoplasm of the
epithelium and cytoplasm of the distal tubule with Con A, BSA-I, WGA and MAA at
different observation days (see table 19). From the infected group no data was
collected because all the kidneys were for NDV by IHC negative.
106
3.4.5 Controls
All samples collected for uninfected controls at our immuno and lectin histochemical
investigations showed no reactions. Regarding the controls for the sugar specificity,
no reaction was observed when the samples were incubated with the respective
inhibitory monosaccharides. To test the specificity of MAA neuraminidase pre-
treatment of the sections was performed. No lectin reactivity was observed after this
treatment. These results demonstrate the specificity of each lectin used in the
present study.
107
Tabl
e 18
. Lec
tin h
isto
chem
ical
resu
lts o
f the
imm
une
syst
em o
f uni
nfec
ted
chic
ken
embr
yos
of d
iffer
ent d
ays
of in
cuba
tion
Lect
inC
onA
BS
A-I
WG
AM
AA
Incu
batio
n da
ys
Org
an
Stru
ctur
e 11
1315
1719
1113
1517
1911
1315
1719
1113
1517
19co
rtex
sd=1
sd=1
2) sd=1
sd=1
sd=1
T H Y M U S
med
ulla
sd=1
sd=1
2) sd=1
sd=1
sd=1
red
Pul
p
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
S P L E E N
whi
te P
ulp
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
part
apic
al
of th
e ep
ithel
ial
cells
5) sd=1
5)
sd=2
sd=1
3)4) sd
=1
4), 5
)4)
, 5)
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
cyto
plas
mof
the
epith
elia
lce
llssd
=1sd
=1sd
=1sd
=1sd
=1
B U R S A F A B R I C I U S
folli
cles
sd=1
sd=1
sd=1
sd=1
4)5)
no
stai
ning
(ave
rage
0)
lo
w s
tain
ing
(ave
rage
1)
m
ediu
m s
tain
ing
(ave
rage
2)
s
trong
sta
inin
g (a
vera
ge 3
)
ver
y st
rong
sta
inin
g (a
vera
ge 4
).
sd=
stan
dard
dev
iatio
n w
ithin
the
sam
e gr
oup,
*=
no d
ata,
1)=
sign
ifica
nt d
iffer
ence
with
11th
i.d.
, 2) =
sig
nific
ant d
iffer
ence
with
13th
i.d.
, 3) =
sig
nific
ant
diffe
renc
e w
ith 1
5th i.
d., 4)
= s
igni
fican
t diff
eren
ce w
ith 1
7th i.
d., 5)
= s
igni
fican
t diff
eren
ce w
ith 1
9th i.
d.
Lect
inC
onA
BS
A-I
WG
AM
AA
Incu
batio
n da
ys
S
truct
ure
1113
1517
1911
1315
1719
1113
1517
1911
1315
1719
Gro
mer
uli
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
5)
sd=1
Api
cal p
art o
f the
ep
ithel
ium
of t
he
prox
imal
tubu
li sd
=1sd
=1sd
=1sd
=1sd
=1
2), 3
)
sd=1
4)4)
sd=1
sd=1
sd=1
sd=1
sd=1
Cyt
opla
sm o
f the
ep
ithel
ium
of t
he
prox
imal
tubu
li sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1A
pica
l par
t of t
he
epith
eliu
m o
f the
di
stal
tubu
li sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1sd
=1C
ytop
lasm
of t
he
epith
eliu
m o
f the
di
stal
tubu
li sd
=1
3) sd=1
sd=1
3) sd=1
sd=1
sd=1
sd=1
sd=1
sd=1
no
stai
ning
(ave
rage
0)
lo
w s
tain
ing
(ave
rage
1)
m
ediu
m s
tain
ing
(ave
rage
2)
s
trong
sta
inin
g (a
vera
ge 3
)
ver
y st
rong
sta
inin
g (a
vera
ge 4
).
sd=
stan
dard
dev
iatio
n w
ithin
the
sam
e gr
oup,
*=
no d
ata,
1)=
sign
ifica
nt d
iffer
ence
with
11th
i.d.
, 2) =
sig
nific
ant d
iffer
ence
with
13th
i.d.
, 3) =
sig
nific
ant
diffe
renc
e w
ith 1
5th i.
d., 4)
= s
igni
fican
t diff
eren
ce w
ith 1
7th i.
d., 5)
= s
igni
fican
t diff
eren
ce w
ith 1
9th i.
d.
108
Tabl
e 19
. Lec
tin h
isto
chem
ical
resu
lts o
f the
kid
ney
of u
ninf
ecte
d ch
icke
n em
bryo
s of
diff
eren
t day
s of
incu
batio
n
109
V. Discussion
1 Determination of the LD50 of the NDV
According to the results observed in the determination of the LD50 in chicken
embryos, the use of the Herts 33-NDV pathotype was discarded in our study (chapter
4, Material and Methods). Due to the fact that it is a very pathogenic virus and the
embryos died too early after infection (average 1.5 days) or were negative by HA-
Test (less than 30% of the surviving infected chicken were positive). The NDV-Herts
33 strain is a viscerotropic velogenic pathotype, and causes acute lethal infections
with prominent hemorrhagic lesions in the gut (ALEXANDER, 1998). The use of a
second passage did not minimize its pathogenicity. For this reason, the studies were
conducted only with the other two NDV pathotypes (Komarov and HB-1).
The infection with mesogenic (Komarov) and lentogenic (HB-1) pathotypes is less
lethal; hence, chicken embryos were exposed longer to the viral infection, and it was
possible to conduct our studies on chicken embryos (chapter 4, Material and
Methods). The mesogenic pathotype leads to low mortality, respiratory or/and
neurological symptoms, and the lentogenic pathotype causes mild or unapparent
respiratory infection (ALEXANDER, 1998).
Following replication at the site of entry, virus particles can spread beyond the
primary site of replication to other tissues, after breaching the physical and immune
barriers. Viruses that escape from local defenses to produce a disseminated infection
often do so by entering the bloodstream (hematogenous spread), and they may have
access to almost every tissue in the host. Hematogenous spread begins when newly
replicated particles produced at the entry site are released into the extracellular
110
fluids, which can be taken up by the local lymphatic vascular system (FLINT et al.,
2004).
In the first hours of infection the viral particles are present in the primary infection site
and primary viremia can be found. Thereafter, replication in other organs can be
found. Primary viremia is the presence of progeny virions released into the blood
after initial replication at the site of entry, the concentration of particles during primary
viremia is usually low. The subsequent dissemination is often extensive, releasing
considerably more virions, and its appearance in the blood is called secondary
viremia (FLINT et al., 2004).
For this study, the time of virus exposition was considered as a relevant aspect,
because an important aim was to observe the effect of the viral infection on the
glycosylation pattern of the cells infected, and therefore the examination of the
embryos was performed after completion of the viral cycle. It is known that the
incubation period of the NDV is in average 5 days (RITCHIE, 1995). Based on these
facts, we decided to examine the embryos at day 4 p.i.
2 Studies in chicken embryos
2.1 Macroscopical observations
In chicken embryos at 11th i.d. infected with both pathotypes (Komarov and HB-1),
the presence of skin hemorrhages was observed (Fig.9). Such lesions are not
described in the literature. Hemorrhagic lesions have only been described in
conjunctiva of the lower eyelid, spleen, thymus, bursa of Fabricius, proventriculus,
small intestine and cecum in two weeks old SPF White Leghorns chicken
111
(KOMMERS et al., 2002) and one day SPF White Leghorns chicken (GOHM et al.,
2000); and trachea in one day SPF White Leghorns chicken (GOHM et al., 2000).
Macroscopical observations independent of the skin were not conducted in this
study, because the embryos were fixed completely in the Bouin´s solution.
2.2 Histological observations
Description of the normal structure of the chicken embryos is an important tool to
determine histological changes in the chicken embryos after infection. A problem in
the present study, in some cases, was the lack of literature with a detailed description
of the histological development of the chicken organs. In most of the cases only
macroscopical descriptions were found. Therefore, the findings of our investigations
are discussed in the context of the literature concerning normal embryonic
development of the chicken.
2.2.1 Respiratory system
In contrast to the mammalian respiratory system, the respiratory system of birds
contains a more simple larynx, syrinx, compact spongy lung and air sacs (PLOPPER
and ADAMS, 2006). In the present study of the respiratory system the trachea, lungs
and air sacs were evaluated.
In the trachea of adult chicken, the lining epithelium is of the respiratory type,
containing ciliated cells, brush cells, goblet cells, basal cells and neuroendocrine
cells. A variety of migratory cells is also observed in the epithelium; these include
lymphocytes, globule leukocytes and mast cells. The tracheal propria and
submucosa consist of loose connective tissue and a subepithelial layer of
longitudinally oriented at elastic fibers, fibrocytes, lymphocytes, plasma cells,
leukocytes and mast cells. Numerous intraepithelial mucous glands and a complete
112
hyaline cartilaginous ring are observed. The mucous cells generally secrete sulfated
acid glycoproteins (PLOPPER and ADAMS, 2006).
The origin of the trachea is observable in the laryngotracheal groove, which appears
at 72 hour of incubation; at the end of the 4th i.d., the trachea starts differentiation
beginning at the posterior portion of the laryngotracheal groove (ROMANOFF, 1960).
Nevertheless, SAKIYAMA et al. (2000) describe this process at 3rd i.d. On the sixth
i.d., the trachea is a long epithelial tube with thick walls at its posterior end
(ROMANOFF, 1960). Histological description of the trachea development has not
been described in the literature, according to our knowledge. In the present study a
detailed description of the chick trachea development were realized at 11th, 13th, 15th,
17th and 19th i.d. (Figure 11).
The lungs comprise primary, secondary and tertiary bronchi (parabronchi), atria and
air capillaries (PLOPPER and ADAMS, 2006). In the present study, parabronchi, atria
and air capillaries were evaluated.
SAKIYAMA et al. (2000) divide lung formation into three parts: 1- Formation of the
respiratory rudiment (2 and 4 days of incubation); 2- the bronchial branching (5th i.d.)
and 3- the formation of the air sacs (6th i.d.). At 5th-6th i.d., the main bronchi initiated
branch to form the secondary bronchi. At 8th i.d., the air sacs can be recognized by
their morphology.
The lungs first arise as diverticula from the foregut at a level posterior to the fourth
visceral pouch and just ventral to the esophagus. The lungs are attached to the
laryngotracheal groove by their ventral surface, and appear first during the third
incubation day. The recurrent bronchi are formed on the ninth day as buds from the
proximal ends of the abdominal and the posterior intermediate air sacs, and later
from the other air sac (ROMANOFF, 1960).
113
The parabronchi are the last part of the tube system of the lung. Their development
from the tenth i.d. consists principally of the establishment of the anastomosing
network and the resultant bronchial circuit. On the twelfth i.d., the parabronchial tips
are almost in direct contact and complete union is achieved by the 15th i.d. After the
18th i.d., further connections are established between parabronchi in other parts of
the lung (internally and externally) (ROMANOFF, 1960).
In the adult chick, the epithelium of the parabronchi varies from the respiratory type
to simple cuboidal or squamous. Numerous small air spaces (atria) open into the
parabronchi (PLOPPER and ADAMS, 2006). The cuboidal epithelium type, as
surrounded by the arranged parenchyma and blood cells was observed in chick lung
at 10th i.d. (SAKIYAMA et al., 2000). Later histological observations of the lung
development are not described in the literature that supported the results (Fig. 12) of
the present study.
The ultimate branches are the air capillaries. After the ninth i.d., the parenchyma of
the lung is arranged in columns around the parabronchi. Minute branches project
from the parabronchi into the lung parenchyma; these branches are the air capillaries
which appear between the 14th and 16th i.d. Between the 19th and 20th i.d., the air
capillaries anastomose, forming an intricate network completely enveloping the
parabronchi (ROMANOFF, 1960). These capillaries show simple squamous
epithelium lines in the fully developed chick (PLOPPER and ADAMS, 2006), the
same aspect as described in our study (Fig.12).
The first air sacs appear at 6th i.d. in the chicken embryo, and further enlargement
occurs at 9th. By the 12th to 15th i.d., the changes in most of the air sacs are growth
related (ROMANOFF, 1960). In the fully developed chick, the terminal air sacs are
lined by a simple squamous to cuboidal epithelium (PLOPPER and ADAMS, 2006).
These descriptions support the results obtained in the present study (Pag. 84).
114
2.2.2 Digestive system
Regarding the digestive system, esophagus, proventriculus, ventriculus, intestine and
liver were evaluated in the present study. Their histological and functional
development is described by ROMANOFF (1960) and supports our data.
In the adult chicken, the esophagus is characterized by a thick, keratinized, stratified
squamous epithelium. The lamina propria consists of loose connective tissue
containing large mucous glands, and the lamina muscularis mucosae is a thin
longitudinally oriented smooth muscle. The submucosa consists of a thin layer of
connective tissue, and the tunica muscularis is composed of an inner circular and an
outer longitudinal layer (FRAPPIER, 2006).
The only portions of the esophagus that have derived from the endoderm of the
foregut are there stratified squamous epithelium and the mucous glands. The more
peripheral portion consisting of connective tissue and muscle derives from the
splanchnic mesoderm (ROMANOFF, 1960).
In the 3th i.d., the esophagus of the chicken embryo is round in cross section and
more than one layer of nuclei is observed in the epithelium. In the epithelium during
the 4th i.d., the nuclei start to divide actively and two or three layers of nuclei are
observed, at this time the esophagus is occluded. The cavity of the esophagus
begins to reestablish itself from posterior to anterior during the 7th i.d. and ends at 8th
i.d. or 9th i.d. The epithelial layers show no great increase in number during
development, only during the last 24 hours before hatching the number of epithelial
cell layers increases very rapidly from ten to twenty (ROMANOFF, 1960), this support
the observation of the present study (Fig.13).
The mucous glands of the esophagus develop from epithelial buds that project into
the lamina propria. These buds appear when development is completed with 60 to
65%. In the chicken embryo at 18th i.d., the buds exhibit secondary projections, and
115
lumina are beginning to develop within them while accumulation of mucinous fluid is
observed. At 19th i.d., the glands are formed with cylindrical cells, containing granules
of premucin or mucinogen (Fig.18; ROMANOFF, 1960), same to the observations
realized in the present study.
The differentiation of the mesodermal portion of the esophagus begins on the 4th i.d.
to 7th i.d. The outer longitudinal layer is visible in the 9th i.d. By the medium part of
incubation time, the lamina muscularis can be identified. As the tunica propria grows
thicker, the lamina muscularis mucosae gradually assumes a position between the
epithelium and the circular muscle layer of the tunica muscularis. In the second half
of incubation, the thickness of the muscle layers increases (ROMANOFF, 1960), this
supports our observations (Fig.13).
The birds do not have a glandular stomach similar to mammals. Instead, they have a
proventriculus (glandular stomach) and a ventriculus (muscular stomach). In adult
chicken, the mucosa of the proventriculus is characterized by papillae with numerous
plicae which are covered by a simple columnar epithelium. It shows proventricular
glands with a simple cuboidal to low columnar epithelium. The lamina propria is
typically made of loose connective tissue, and a typical tunica muscularis and tunica
serosa are present (FRAPPIER, 2006).
116
Figure 18. Mucous gland development in the esophagus of the chick embryo (ROMANOFF, 1960, modified) A. 16 incubation day embryo, B. 18 incubation day embryo, C. 19 incubation day embryo.1. esophageal epithelium, 2. solid epithelial bud, 3. vacuoles, 4. lumen of gland.
On the 6th i.d. or 7th i.d., a semistratified cylindrical epithelium is observed in the
compound glands of the proventriculus. After 9th i.d. to 11th i.d., a single layer of
cylindrical cells is observed. By the end of the 11th i.d., the glands have given off
buds basally, the cylindrical epithelium decreases to cuboidal as the gland becomes
multilobular, and they are extended throughout most of the tunica propria. After the
15th i.d., the principal change is a rapid increase in the number of secondary lobules
or saccules. Granular, argentophilic cells appear in the superficial epithelium of the
proventriculus at 8th i.d. and increase in number until 13th i.d. After 16th i.d., they
117
disappear (Fig. 19; ROMANOFF, 1960), supporting our observations regarding
proventriculus.
Figure 19. Diagram of successive stages in the structural and cytological development of the proventriculus of the embryo chick (ROMANOFF, 1960; modified)A. 9 incubation day, B. 10 incubation day, C. 11 incubation day, D. 12 incubation day, E. 13 incubation day, F. 14 incubation day, G. 18 incubation day, H. at hatching.1. alveolus of gland, 2. secondary duct of the gland, 3. primary duct of gland, 4. orifice of gland, 5. submucosa, 6. lamina mucosae, 7. circular muscle,8. longitudinal muscle, 9. mucous gland.
In adult chicken, the ventricular surface of the epithelium is simple columnar with
tubular mucosal gland, which show a simple cuboidal epithelium. The lamina propria
and the tela submucosa are composed of loose connective tissue; the lamina
muscularis mucosae is very discontinuous. The tunica muscularis consist of a single
118
thick layer of smooth muscle cells, and the organ also shows a typical tunica serosa
(FRAPPIER, 2006).
The ventriculus is outstandingly different from the remainder of the digestive tract,
because the glandular and mucosal layers are relatively thin and the circular
musculature is massively developed. The tunica muscularis is visible at 7th i.d., and at
11th i.d. the circular layer is thicker, the lamina propria is thin and dense, and the tela
submucosa inconspicuous (ROMANOFF, 1960).
The tubular ventricular glands appear at 5th to 9th i.d., when the high cylindrical
epithelial cells in the ventriculus multiply, so that 2 or 3 rows of nuclei increase to 4 or
5 and initiate mucin secretion. At the 10th and 11th i.d., secretion increases, while at
12th i.d. an increase of cell number is observed, and they project into the ventricular
lumen assuming the appearance of tubular glands. The final stage is observed at 18th
i.d., when the secretion of keratinoid substances begins deep in the fundi of the gland
(Fig. 20; ROMANOFF, 1960). Similar observations were realized in the present study
(Fig. 14).
The small intestine is divided into three parts: the duodenum, jejunum and ileum. In
adult chicken, the general histological structure of the small intestine presents as first
layer the tunica mucosa consisting of a simple columnar epithelium, which contains
numerous goblet cells interspersed among the epithelium, the lamina propria with
crypts of Lieberkühn. The next layer observed is the tela submucosa; it is formed by
connective tissue. The lamina propria and the tela submucosa contain large amounts
of diffuse and nodular lymphatic tissue. The tunica muscularis is composed of inner
circular and outer longitudinal layers of smooth muscle cells, and the outermost
tunica is typical serosa (FRAPPIER, 2006).
119
Figure 20. Stages in the development of glands of the ventriculus lining in the chick embryo (ROMANOFF, 1960; modified) A. 11 incubation day, B. 13 incubation day, C. 17 incubation day, D. 18 incubation day.1. epithelium, 2. extracellular process, 3. layer of secretion, 4. dark line of granules, 5. cellular inclusions of mucin, 6. strands of cells, 7. layer of mucin, 8. degenerating cells, 9. keratinoid substance.
The small intestine is lined with simple columnar epithelium at 3th i.d., and during the
4th i.d., the epithelium becomes pseudostratified and an increase of the mitotic
activity is observed. The duodenal folds appear first at 8th i.d., and they extend to the
end of the small intestine at 11th i.d. Its number increases through embryonic
development. Villus formation begins at the 13th i.d., and at the 16th i.d. vascular
tissue invades the villi, and the epithelial cells are now cuboidal. During the last days,
the villi elongate and the epithelium is again approaching the columnar form. Crypts
of Lieberkühn appear first at 14th i.d. and goblet cells do not appear in abundance
120
until late in development. Round cells with eosinophilic granules are present in the
duodenal epithelium at 15th i.d. (ROMANOFF, 1960).
The differentiation of the mesenchymal portion of the intestine begins on the 5th i.d.,
when the mucosa can be distinguished and undifferentiated myoblasts are disposed
in a circular and longitudinal layer. The lamina muscularis mucosa is present on the
14th i.d., this muscle layer starts to extend into the intestinal villi at 17th i.d.
(ROMANOFF, 1960).
The large intestine is composed of the cecum, colon, rectum and the cloaca. In the
present study only the cecum was evaluated. Two ceca open into the digestive tract
at the junction of ileum und rectum. They are divided in three portions: The proximal
portion contains prominent villi, the middle portion shows shorter and broader villi and
mucosal folds, and the distal portion is devoid of villi (FRAPPIER, 2006). The other
histological structures are similar to those described for the small intestine.
In the large intestine, mitotic activity is observed in the epithelial cells at day 4th i.d.,
and at 6th i.d. the mesenchyme is in its initial stages of differentiation into muscular
and submucosal layers. At the twelfth day, cylindrical epithelium is observed. At the
14th i.d., great numbers of crypts of Lieberkühn appear. The formation of villus begins
at 18th i.d., they are lower, thinner and less numerous than those in the small
intestine (ROMANOFF, 1960). On the 14th i.d., argentaffin cells appear and goblet
cells are present by the end of incubation. The circular muscle layer of the tunica
muscularis appears at 7th i.d. and the longitudinal layer is present on the 11th i.d.
(ROMANOFF, 1960). The knowledge from the literature supports the results obtained
in the present study regarding the structural development of the intestine of the
chicken embryo (Fig. 15 and 16).
Regarding the histology of the adult liver, each lobe of the liver is covered by a typical
tunica serosa. Between the lobes, interlobular connective tissue is present containing
the vascular and bile duct systems. Smooth muscle cells may be present in the
121
capsule and the interlobular connective tissue. The parenchyma is composed of
hepatocytes. They are characterized by a centrally located spherical nucleus, with
one or more prominent nucleoli and scattered clumps of heterochromatin, the
cytoplasm varies depending on nutritional and functional changes (FRAPPIER,
2006).
The liver parenchyma at 4th i.d. consists of tubules, each with a small lumen.
Sinusoids are narrow of beginning, but they gradually become wider until the 5th or
6th i.d. At 9th i.d., the sinusoids decrease in diameter as the liver parenchyma
increases in density. The hepatocytes increase in size at 12th i.d., probably correlated
with the accumulation of lipoid inclusions in the cytoplasm (ROMANOFF, 1960). In
the present study, the structure of the liver observed was in accordance to that
described by ROMANOFF (1960) and FRAPPIER (2006), but, in contrast, presence
of vacuoles (11th i.d.) and lipoid deposits were not observed before the end of the
incubation time (17th i.d.).
2.2.3. Immune system
In post hatching chicken, the thymus consists of lobules surrounded by connective
tissue. Each lobule is composed of a cortex and medulla. The thymic cortex consists
of an epithelial reticulum and lymphocytes. The epithelial reticular cells have large,
pale, ovoid nuclei and long branching cytoplasmic processes that contain numerous
intermediated filaments. This cortex stains darker than the medulla because it
contains a large number of lymphocytes. The medulla is composed also of epithelial
reticular cells; many of them are similar to those observed in the cortex but others are
much longer, and some of these cells form thymic corpuscles (Hassall`s corpuscles).
The corpuscles consist of one to several calcified or degenerated large central cells
surrounded by flat keratinized cells in concentric arrangement. Interdigitating
dendritic cells are also present in the medulla (PRESS and LANDSVERK, 2006).
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The thymus originates from the endoderm of the third pharyngeal pouch (PRESS and
LANDSVERK, 2006). It is visible first as a rudiment at the 5th i.d., looking like a mass
of epithelial cells, which elongates to form an epithelial cord extending along the
jugular vein. At the end of the 7th i.d., the thymus is syncytial and appears reticular.
By the 11th, i.d. the first blood vessels enter the thymus and small thymic cells appear
in the gland. Eosinophil cells appear in large number about the 16th i.d. At the end of
the 17th i.d. the thymus is well defined into zones, and at the 19th i.d., Hassell´s
corpuscles are acidophilic (ROMANOFF, 1960). The histological structure observed
in the present study is in accordance to the described by the latter author,
nevertheless eosinophil cells were not observed.
In adult chicken, the spleen is surrounded by a thick connective tissue capsule.
Trabeculae composed by collagen and elastic fibers, as well as smooth muscle cells
extend from the capsule into the parenchyma. The trabeculae contain arteries, veins,
lymph vessels and nerves. The parenchyma is composed of a red and a white pulp.
The red pulp consists of venous and splenic cords (vascular channels lined with
elongated, longitudinally oriented endothelial cells). The white pulp is lymphatic
tissue that is distributed throughout the spleen and is comprised of lymphatic nodules
and diffuse lymphatic tissue called periarterial lymphatic sheaths (PALS). Nodules of
the white pulp are B-cell zones. Throughout the white pulp, reticular cells and
associated reticular fibers containing lymphocytes, macrophages and dendritic cells
are present (PRESS and LANDSVERK, 2006).
The spleen is of mesodermal origin, and it appears firstly at 4th i.d. as a condensation
of cells. It presents intense multiplication at the 6th or 8th i.d. and starts to acquire the
spongy structure characteristic of the red pulp. On the 12th i.d., arterial vessels start
to appear in the spleen. Mesenchymal cells proliferate intensively around the arteries
and their small rami by the 15th i.d. Between 15th and 17th i.d. small lymphocytes
begin to form the white pulp (ROMANOFF, 1960). In contrast, in the present study a
differentiation of white and red pulp was not observed; however, all the cells
described were present.
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The bursa of Fabricius is considered to be functionally equivalent to the mammalian
bone marrow in regard to the differentiation of B cells (PRESS and LANDSVERK,
2006). The primordium of the bursa of Fabricius appears in the form of a median
lamina of endodermal epithelium, and proliferates dorsally and caudally from the anal
plate at 6th i.d. Buds of thickened epithelium project into the mucosal layer. These
buds begin to appear on the 12th i.d. Some of the persisting epithelial cells align
themselves in a single row around the periphery of the bud (basal membrane), while
others are transformed into stellate cells and form the reticular framework of the
medulla. Invasion of lymphocytes is common from the 14th to 18th i.d. (ROMANOFF,
1960).
These observations support the data obtained in the present study. PRESS and
LANDSVERK (2006) described migration of precursor cells committed to the B-cells
lineage from 8th to 15th i.d. of chicken embryo development. Nevertheless, a
considerable number of granulocytes was observed also in the present study in the
connective tissue of the bursa of Fabricius (Fig.17). This phenomenon was observed
in the control and the infected group, and for this reason it is considered as normal
during the development of the chicken bursa.
2.2.3 Urinary system
The excretory system appears early in embryonic development arising from the
intermediate mesoderm. Its development proceeds in three stages: pronephros (it is
of short duration and disappears almost completely by the fourth incubation time),
mesonephros (its function begins at the fifth day and continues until the eleventh
incubation day) and metanephros (appears on the fourth i.d., but its function is not
initiated before the development of the mesonephros has finish).
In the chicken embryo, the renal corpuscle is composed of a round to elliptical and
relatively large glomerulum that appears first at 4th i.d.; the capsule is composed of
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connective tissue. The mesonephric tubules arise as condensation of cell masses
and posterior show a cuboidal epithelium. By the 11th i.d., the blood supply increases
greatly, extending to the peripheral metanephrogenic tissue and more abundantly to
the tubules and glomeruli (ROMANOFF, 1960). This explains the presence of great
numbers of erythrocytes present into the nephronic tissue observed in the present
study. From the 13th to 18th i.d. the number of the differentiated tubules increases
(ROMANOFF, 1960). All the structures described in the literature are observed also
in the kidney in our study.
2.3 Histopathological observations
Regarding the histopathologic changes in NDV infected chicken embryos, hypoplasia
of epithelial cells and the absence of cilia and goblet cells in the trachea were
described, all other organs evaluated showed no changes.
These results are supported by the findings from chicken after vaccination against
NDV, at the level of the tracheal epithelium, where an almost complete deciliation of
the epithelial surface was observed, because the original pseudostratified epithelium
was replaced by a simple squamous to cuboidal epithelium (MAST et al., 2005). In
contrast, epithelial hyperplasia with necrosis and fibrin deposits in the trachea was
described in chicken infected with NDV (KOMMERS et al., 2002).
Changes in the cellular composition of the surface epithelium can be a direct
consequence of ongoing injury (HONG et al., 2004). After crossing the epithelium,
virus particles reach the basement membrane. The integrity of which may be
compromised by epithelial cell destruction and inflammation (FLINT et al., 2004).
Other histopathological changes observed in chicken infected with NDV are necrosis
of cardiac myofibers, spleen, bursa of Fabricius, thymus, pancreas and liver.
Necrosis and hemorrhages within the lamina propria were described also for infected
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chicken regarding the proventriculus and small intestine (KOMMERS et al., 2002). In
SPF chicken experimentally infected with mesogenic pathotype splenic lymphoid
hyperplasia, myocardial inflammation with degeneration of myofibers and infiltration
of macrophages has been described. Whereas, when they were infected with
lentogenic pathotype, splenic lymphoid hyperplasia and prominent lymphoid follicles
in air sacs were observed (BROWN et al., 1999).
The presence of mononuclear inflammatory cells (lymphocytes, plasma cells and
macrophages) has also been described as histopathologic changes in tissues
(KOMMERS et al., 2002). In the present study, these cells were not observed, but
granulocytes and erythrocytes were found in different tissues, both in the control
group and the infected group. Other authors have described that these cells are
normally present during chicken development (ROMANOFF, 1960).
2.4 Histochemical observations
2.4.1 Immuno histochemical observations
Epithelia from trachea, lung, air sac, esophagus, ventriculus and proventriculus were
positive for IHC (more than 50%) in both infected groups (Komarov and HB-1) at 11th
i.d. (Table 13). These findings are the similar as those of KOMMERS et al. (2002);
however, they found positive results also for thymus, spleen, bursa of Fabricius,
small intestines, cecum and kidney by using the same technique in chicken infected
with NDV.
Presence of NDV, detected by PCR, has been reported in the trachea, lung and
cecal tonsil of SPF chicken inoculated at 3 weeks of age with LaSota pathotype.
Kidney, spleen and proventriculus were not detected (PEROZO et al., 2006). In
chicken of an age of 4 weeks inoculated with the NDV-99 299 pathotype, the
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presence of the virus determined by PCR was noticed in the trachea, lung, thymus,
kidney, spleen, liver, intestine and bursa of Fabricius (BARBEZANGE et al., 2002).
Spleen and heart of SPF chicken infected with the mesogenic pathotype showed
positive IHC reactions, and air sac and heart were also positive after infection with
the lentogenic pathotype. It is important to consider that the reaction in this case was
inconsistent, but the infection was performed via the conjuntival sac (BROWN et al.,
1999).
The type of infected organs is dependent of the pathotype present (HUANG et al.,
2004). Nevertheless, in the present study no differences between the pathotypes
used were observed in this regard (Table 13).
Persistent infection of embryonic chicken tracheal organ cultures with Newcastle
disease virus (NDV) is described. Tracheal explants remained morphologically intact
and were able to support the replication of NDV for 6 months. Peak titer of released
virus occurred at 1 week post-infection, whereas maximal immuno fluorescence was
not observed until 30 days post-infection. By electron microscopy and immuno
fluorescence are observed that the cells of the subepithelial connective tissue as the
site of NDV persistence (CUMMISKEY et al., 1973).
The technique used for diagnosis and the time of exposition to the virus can play an
important role in the determination of the virus distribution. During the last years, the
development of PCR techniques has been extended for the diagnosis of different
diseases, also for NDV (LI and ZHANG, 2004). In some cases they are used to
determine the presence of virus in tissues (LI and ZHANG, 2004), but when
macerated tissue is used for extraction, the virus may be present in the blood vessels
of the organs, and not in the cells forming the organ. For this reason, in the present
study histochemical techniques were used. They can help to elucidate the
pathogenesis of viral infection.
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Virions can be released from the apical surface of cells, from the basolateral surface,
or from both. After replication, particles released from the apical surface are back
where they started, outside the host. In contrast, virus released from the basolateral
surfaces of the polarized epithelial cells has been moved away from the defenses of
the luminal surface (FLINT et al., 2004).
In general, virions released at apical membranes establish a localized or limited
infection, where local lateral spread from cell to cell occurs in the infected epithelium,
but virions rarely invade the underlying lymphatic and circulatory vessels (FLINT et
al., 2004). They can be the reason why in our study the presence of the NDV was
detected only in epithelial cell of the infected organs, and absence of NDV was
observed in blood vessels or disseminated in the tissues evaluated (Fig.10).
Differences between results using the HA-Test and IHC were observed in the group
infected at 15th i.d. Almost all the embryos which were positive in the HA-Test at this
time were negative by IHC testing (Table 13). The embryonated chicken eggs have
long been one of the most widely used host system for the isolation, propagation and
characterization of avian viruses. The embryo and its supporting membranes provide
the diversity of cell types necessary to culture many different types of viruses
(SENNE, 1998).
In order to determine if the virus was present in the HA-Test negative chicken
embryos, tissues of some of these infected at 11th (n=2) and 15th (n=2) i.d., were also
tested by IHC. Embryos infected at 15th were positive in IHC, but these phenomena
were not observed in embryos infected at 11th i.d., whereby animals negative by HA-
test were also negative to IHC (data not shown). It is possible that the virus still
remains in the membranes and no replication in the embryo is developed. It is
important to remember that by HA and IHA-testing the presence of the virus was
detected in the allantoic fluid.
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This phenomenon may be explained by the presence of glycoconjugates in the
embryonal membranes. Reactions with the lectins WGA and MAA in the CAM
epithelia were present in the embryonal chicken membranes, and the sites and
intensity of the lectin binding gradually increased up to hatching (GABRIELLI et al.,
2003). It is possible that the virus is still present in the egg and replicates only in the
membrane, and for this reason the HA-test (allantoic fluid) was positive although the
virus still was not present in the embryonic tissues. Perhaps an evaluation of the
embryonal membrane can help to elucidate these aspects.
Another possible explanation of this result is the presence of endogenous lectins that
are present in chicken during organogenesis, for example galectins in the kidney
(STIERSTORFER et al., 2000), liver and intestine (LIPS et al., 1999). These lectins
are involved in several extra-and intracellular functions and are described as
important tools for the innate immune response. It is known, that the presence of
galectin-reactive proteins indicate gradual increases in liver and intestine size during
the developmental stages of the chicken (LIPS et al., 1999).
Another important endolectin that might have produced interference with the viral
replication in this case is mannose-binding lectin (MBL). It is a C-type lectin that plays
a crucial role in the activation of the lectin pathway; which is involved in the first line
of host defense against pathogens (SEKINE et al., 2001; FUJITA et al., 2004;
TIZARD, 2008). Its presence was also reported in chicken (LYNCH et al., 2005).
Measurements of the level and distribution of MBL were described in different tissues
during embryogenesis and through early and adult life (LAURSEN et al., 1998;
NIELSEN et al., 1998). The level in egg yolk is comparable to that in serum and it is
considered as maternal chMBL, and, like maternal antibodies, it is transported from
the yolk sac to the embryo (LAURSEN et al., 1998).
Developing avian embryos are transiently protected against bacterial toxins, bacteria,
parasites and viruses by maternal Ig transferred via the yolk (FELLAH et al., 2008). It
is also possible, that the embryo takes up proteinaceous substances in the process
129
of drinking amniotic fluid which interfere with the virus replication. Drinking amniotic
fluid process begins in the embryonic chicken at 13th day of incubation. Nevertheless,
proteins have been found in the yolk, blood and amniotic fluid from the fifth day
(FREEMAN and VINCE, 1974).
2.4.2 Lectin histochemical observations
When considering the biological significance of the role of glycosylation patterns in
virus infection, the cell surface is the most important structure for virus entry into the
cell. It is known that in paramyxoviruses fusion with cell membranes occurs at the cell
surfaces; in contrast with Influenza virus, where fusion takes place in the cytoplasm
(BULLOUGH et al., 1994). In addition, virus was detected by IHC only in the
epithelial cells of the different organs (trachea, lung, air sac, esophagus, ventriculus
and proventriculus). For this reason, this part of the discussion will focus on the
observations of the epithelial cells of the different organs evaluated.
Significant differences (Fisher Exact test �0.05) of glycosylation patterns were
observed with Con A, BSA-I, WGA and MAA in the trachea, lung, air sac, esophagus,
proventriculus, ventriculus, large intestine and Bursa of Fabricius in the control group
as compared with the different i.d. evaluated. No relations between the differences of
the glycosylation pattern in the control group (between the different i.d./ 11 and 15)
and virus infection were detected (Table 14, 16 und 18).
Regarding glycosylation pattern of the organs that were negative or less than 40%
positivity by IHC in this study, the liver showed weak reactions with all used lectins
(Table 16). Similar results were reported by FELDMANN et al. (2000) from chicken
liver using MAA. In contrast, in the mouse, liver imprints fixed in acetic acid-ethanol
solution showed strongest Con A labeling of hepatocytes (MORAES and MELLO,
2006).
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Different glycosylation patterns in the intestine were observed with all the lectins
used in this study, according to the cell types observed, the incubation time of the
chicken embryo and the animal observed (Table 16). According to those
observations it is possible to assume that differences of sugar moieties are present in
this organ. This could have an influence on the IHC reaction, because the rabbit
antiserum against NDV was polyclonal, so that unspecific reactions cannot be
excluded. This hypothesis requires further investigation and more studies of the
glycosylation pattern with other lectins have to be made. Unspecific reactions due to
the detection system are unlikely, because the system used in the study does not
react with alkaline phosphatases or with avidin.
In mice, the reactivity with WGA in intestinal goblet cells was strong, and with Con A
and BSA-I a weak reaction was observed (KANDORI et al., 1996). In contrast to the
present study, strong reaction was found in all cell type in the intestinal epithelium
with Con A, BSA-I and WGA (Table 16). These results agree with those reported for
chicken in the age of 2 days, 15 days and 30 days, where strong reactions was found
with Con A and WGA in the epithelium of the jejunum and cecum (POHLMEYER,
2002).
In the rat colon, presence of sialoglycoconjugates was observed, and the occurrence
and distribution of sialic acids linked α2,6 to D-Gal/D-GalNAc and α2,3 to D-Gal were
directly demonstrated with SNA and MAL II binding, respectively (ACCILI et al.,
2008).
The observations made in the thymus (Table 18) are similar to those reported for the
chicken embryo by FERNANDEZ et al. (1994) using WGA and Con A. These authors
had examined the microenvironment of the chick thymus during development using
WGA, Con A, RCA-I and TPA on thymic sections from 13th, 15th, 17th and 19th i.d.
chick embryos. The authors observed that Con A lectin detected several cell clusters
of stromal cells and thymocytes in cortical regions. The sugar residues detected by
RCA were distributed both in stromal cells and thymocytes of the developing chick
131
thymus. There was an increase of the reaction intensity to RCA between the 19th i.d.
embryos. The thymic stromal cells stained with immunoperoxidase conjugated TPA
showed a reticular pattern in the medulla (FERNANDEZ et al., 1994). It has also
been reported that Con A and WGA moderately labeled the T-lymphocytes in the
cortex in 2 day old chicken. WGA labelled both the cortical and medullary thymic
stroma at all the stages analyzed (JÖRNS et al., 2003).
An upregulated biosynthesis of O-glycosylation linked glycans on T cell surface
glycoproteins are reported, suggesting that the modification of GalNAc transferase
activity plays a relevant role during the maturation process of thymic cells. In this study,
they analyzed O-glycosylation in thymocytes from mice (control and dexamethasone-
treated) by using PNA and ALL. The authors found that GalNAc transferase activity was
six-fold higher in thymocytes from control mice than from dexamethasone-treated mice;
the rate of diglycosylated peptides for dexamethosone-resistant ALL+ was two fold
higher than for ALL- thymocytes (ALVAREZ et al., 2006). For this reason it may be
that the sugar moiety reactions observed in the present study were weak. Nevertheless,
it has been demonstrated that differentiation and maturation of the thymocytes
generally include changes of the glycosylation pattern of the cell surface (KRISHNA and
VARKI, 1997).
In the present study, differentiations between the zones of the spleen were not
evident and weak reactions with all the lectins used were observed (Table 18). In the
literature it is stated that in 2 day chicken a weak reaction with Con A in spleen cells
and matrices within the B-cell area of the peri-ellipsoid lymphocyte sheaths (PELS)
and T-cells areas occurs, as well as in large mononuclear cells in the red pulp at 2
and 30 days old, other lectins show similar reactions in these structures (Conarva,
LCA, LOA and NPA), demonstrating presence of mannose (JÖRNS et al., 2003).
Staining with WGA was most intensive in comparison with other glcNac-specific
lectins used (CMA, LEA; DSA and LEA) of the B-lymphocytes in the PELS, T-
lymphocytes in the peri-arteriolar lymphocyte sheaths (PALS) and the red pulp, and
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any structure was labeled with MAA in chickens at 2 and 30 days old, but SNA-I
labeled cells and matrices within T-cell and B-cell areas, endothelium and cells with
dendritic morphology in the red pulp (JÖRNS et al., 2003).
In the present study in the bursa of Fabricius strong reactions were observed in the
epithelium and follicles with Con A, BSA-I and WGA (Table 18) and similar results
are reported in the literature (JÖRNS et al., 2003). Con A labeled the B-cells areas,
medulla and follicle associated epithelium in chicken at 2 days old and 30 days old,
and WGA labeled medulla at 2 and 30 days old. In the meanwhile, MAA labeled the
epithelium of the bursa, but JÖRNS et al. (2003) describe this labeling with MAA as
more specifically located in the follicle associated epithelium.
The results observed in the kidney with Con A and WGA (Table 19) are supported by
the literature for other animal species. In the rabbit, strong reaction for the kidney had
been described for Con A and WGA (OJEDA and PIEDRA, 1994; RIELLE et al.,
1987). Regarding the staining of kidney tissue with other lectins used in the present
study, no comparable observations were found in the literature. The distribution of
sialic acid residues in rat kidney urinary tubule was investigated by light and electron
microscopy with LFA. The authors observed intense plasma membrane labeling of
the epithelium of the entire proximal tubule and the thin limbs of loop of Henle. All
cells of the convoluted distal tubules were labeled along their plasma membrane
(ROTH and TAATJES, 1986).
In human fetal kidney, expression of cellular glycoconjugates was studied using
fluorochromelabeled lectins. Each lectin used revealed a characteristic binding
pattern during the phenotypic change of the nephrogenic mesenchyme and during
distinct stages of nephron development. Binding sites for HPA in the renal corpuscle
were also expressed only transiently during nephrogenesis. During further
development, PSA, Con A, WGA and RCA-I reacted with mesangial cells in addition
to the glomerular basement membranes (HOLTHOFER and VIRTANEN, 1987).
133
Regarding to IHC in the respiratory system all organs examined (trachea, lung and
air sac) (Table 13) were positive to this technique. The epithelial cells of these organs
in the control group showed strong or very strong reactions with Con A, BSA-I and
WGA, but only the lung (parabronchi) demonstrated a strong reaction with MAA at
11th and 15th i.d. (Table 14). In the literature, a similar reaction is depicted for Con A
and WGA in the chicken thoracic air sac (BEZUIDENHOUT, 2005), chicken lung
(parabronchi) (GHERI et al., 2000), and with MAA also in the chicken lung
(parabronchi) (FELDMANN et al., 2000).
No reaction with MAA in tracheal epithelial cells was observed by YOON et al.
(1998), but a positive reaction for SNA (Sambucus nigra agglutinin), in mice. This
result is in contrast to the findings of UENO et al., also in mice (1994), and WINTER
et al. (2008) in chicken who describe a strong reaction with MAA in the same tissue.
Our findings for the tracheal epithelium in SPF chicken embryos are in accordance
with the finding of YOON et al. (1998), because no or only a weak reaction was
detected with MAA (Table 14). YOON et al. (1998) consider that these differences
may be due to the use of pre-treatment. It is possible that pre-treatment, for example
with microwave or exposition to high temperatures, change the composition of the
carbohydrate moieties in sialoglycoproteins.
However, SCHULTE and SPICER (1985) report the presence of β-galactose and α-
N-acetylgalactosamine in the tracheal surface epithelium of rats, mice and hamsters,
which was detected by BSA-I and WGA, respectively. These finding support our
results (Table 14), but no references where found for the chicken regarding these
lectins.
In the digestive system of control embryos, strong or very strong reactions were
detected with Con A and WGA in the epithelial cells of the esophagus. In contrast, in
the ventriculus and proventriculus, the reaction intensity was variable (average ±1,
Table 16). At the same time when these reactions where detected in the digestive
system of the controls (i.e., 11th and 15th i.d.), the virus was inoculated in the infected
134
groups. Hence, it is possible to conclude, according to this result, that the presence
of carbohydrates moieties detected by Con A, BSA-I and WGA reactions play a more
relevant role in the infection with NDV, than the moieties detected by MAA.
It is also known that sialic acids are typically found as terminal monosaccharides
attached to cell surface glycoconjugates. They play various important roles in many
physiological and pathological processes, including microbe binding that leads to
infection, regulation of the immune response, the progression and spread of human
malignancies and in certain aspects of human evolution (VARKI and VARKI, 2007).
But in contrast, in the present study, sugar moieties detected by MAA seem not to
play the main role in NDV infection, because for example, organs like kidney and
bursa of Fabricius that showed strong reactions with this lectin, were negative by IHC
(summarized in Fig. 21). Nevertheless, experiments to determine the presence of
other sialic acids were not made, for example by using SNA or other lectins; so the
presence of other receptors might be involved. For example, it has been suggested
that the presence of single sialic acid recognition sites in the paramyxovirus HN
glycoprotein can switch between the function of the binding site and catalytic site
(CONNARIS et al., 2002).
135
0
1
2
3
4
Trachea (EA) Lung (PEA) Air sac (EA) Esophagus(EA)
Ventriculus(EA)
Proventriculus(EA)
Liver (H) Bursa ofFabricius (EA)
Kidney (TDA)
Inte
nsity
of t
he re
actio
n
Con A BSA-1 WGA MAA
Figure 21. Glycosylation pattern of the cells of organs from uninfected chicken embryos at 11th i.d.EA= Apical surface of the epithelial cell, P= Parabronchi, H= Hepatocyte, TDA= apical surface of the epithelium of the distal tubuli.
The analysis of the three-dimensional structure of the NDV-HN protein revealed the
presence of a large pocket, which mediates both activities (receptor binding and
neuraminidase activities). The presence of a second sialic acid binding site on HN
was revealed [thiosialoside neu5Ac-2-S-alpha (2,6) Gal1OMe], and its activity was
confirmed by use of mutant viruses the HNs of which were mutated at Arg516. They
found that the promotion activity of HN was substantially reduced by the mutation
and suggested that the second binding site facilitates virus infection and growth
(KRISHNAMURTHY et al., 2000; BOUSSE et al., 2004). Maybe the use of an
alternative lectin (such SNA-I) may help to elucidate this question.
FERREIRA et al. (2004) consider that NDV requires different sialic acids,
gangliosides and glycoproteins for entry into the cell; they proposed gangliosides as
136
primary receptors while N-linked glycoproteins would function as the second receptor
for viral entry.
Effects of Con A on the adsorption and penetration of the paramyxovirus into cell
cultures of chicken embryos and calf kidney have been described. The authors
concluded that in the cells pre-treated with Con A and infected with NDV more virions
are adsorbed as compared with untreated cells. However, lower viral titer was
observed in cells after pre-treatment (VASILEVA and DUMANOVA, 1976).
A study conducted with chicken erythrocytes to determine the lectin-binding profiles
of NDV, demonstrated that NDV was bound by Con A. Other lectins (SBA, PHA,
DBA, LCA, ASA and LTA) did show not binding patterns that can be associated with
the elution rate (McMILLAN et al., 1985).
All reports support the results of the present study, where mannose seems to play a
more relevant role in virus infection than other carbohydrate moieties. According to
our results, α-D-galactose, α-linked galactose and GalNac can play an important role,
but references that support these results were not found. A virus must come into
contact with the cell and tissues, in which it can replicate. Such cells are normally
recognized by means of a specific virion-cell receptor interaction. The presence of
such receptors determines whether the cell will be susceptible to the virus (FLINT et
al., 2004).
Stronger reaction of lectins were observed in infected organs from the chicken
embryos infected at 11th i.d. Compared to the control group, significant differences
were observed (Fisher´s Exact Test p�0,05) in the epithelium of the trachea with Con
A, BSA-I and epithelium, and goblet cells in the ventriculus with MAA (Table 15, 17
and Fig. 22).
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Con
A c
Con
A c
Con
A c
Con
A c
Con
A c
Con
A c
Con
A c
Con
A i
Con
A i
Con
A i
Con
A i
Con
A i
Con
A i
Con
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BSA-
1 c
BSA-
1 c
BSA-
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BSA-
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WG
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MAA
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0
1
2
3
4
Trachea (EA) Lung (PEA) Air sac (EA) Esophagus(EA)
Ventriculus(EA)
Proventriculus(EA)
Liver (H)
Inte
nsity
of t
he re
actio
n
Figure 22. Glycosylation pattern of the cells of organs from uninfected and infected chicken embryos with Komarov pathotype at 15th i.d.EA= Apical surface of the epithelium, P= Parabronchi, H= Hepatocytes, c= control group, i= infected group.
Some studies have been conducted to determine glycosylation changes in cells
undergoing neoplastic change or deposit diseases and in these cases, increments of
the glycosylation were observed (BROOKS et al., 1997; THÖM et al., 2007). To our
knowledge, lectins are used to observe glycosylation pattern after a viral infection in
order to elucidate the role of carbohydrates moieties as receptor (ARNBERG et al.,
2000; DELPUTTE and NAUWYNCK 2004; WINTER et al., 2006), but this is the first
time that the glycosylation pattern with plant lectins were also observed after viral
infection as change occurring at the cell surface.
Cell injury caused by viral replication may result in visible changes in the cell as CPE.
Because virus-induced CPE are clearly relevant to viral pathogenesis, much effort
has been devoted to understanding how infection alters cell structure and
138
metabolism. Consequences of viral infection may also include cessation of essential
host processes such as translation, DNA and RNA synthesis, and vesicular transport,
but it is possible that its effect has long time post-infection consequences (FLINT et
al., 2004). This effect may alter the production and/or expression of external-
membrane glycoproteins.
Vertebrate cells are covered by a glycocalyx, because the cell membrane is
composed of glycolipids, glycoproteins, glycophospholipids or proteoglycans, and the
biosynthesis of such substances takes place mainly in compartments of the
endoplasmic reticulum-Golgi pathway (DRICKAMER and TAYLOR, 2006). The
expression of these gene products is altered in embryogenesis, cancer, injury and
inflammation, resulting in altered glycan patterns (VARKI and VARKI, 2007). Hence,
it is possible to expect that viral infection induces changes in different cells, in turn,
may have caused different glycosylation patterns that were observed in the present
study (e.g trachea and ventriculus).
3 Conclusions
Precise knowledge regarding the normal histological structure present during chicken
embryo development is important to determine histopathological changes in infected
embryos, in order to rule out the possibility of wrong interpretations resulting from the
presence of different cell types as appear during the developmental period. The
trachea presents histopathological changes independently of the viral pathotype
used, but only in those chicken embryos infected at 11th i.d. Other organs show no
changes after infection. Nevertheless, the absence of histopathological changes did
not indicate the absence of infection.
139
Independent of the pathotype used (Komarov or HB-1), NDV was detected in more
than 50% of the chicken embryos infected at 11th i.d. by IHC in the tracheas, lung, air
sac, esophagus, proventriculus and ventriculus. The virus was detected less than
50% in the liver, thymus, bursa of Fabricius and spleen of the chicken infected at the
same i.d. Only two tracheas, two lungs, one esophagus and one ventriculus were
detected NDV antigen positive in the embryos infected at 15th i.d. On the other hand,
evaluation of the infected embryos which were negative in HA-test is necessary in
order to elucidate the systemic viral distribution in this group. The kidney was
negative in all the animals observed, independently of the experimental group.
IHC is a specific technique to determine the distribution of viral antigen in the above
mentioned organs. However, IHC revealed unspecific reactions under the
experimental conditions employed when the intestine was studied. Also, no
correspondence between HA-test results indicative for the presence or absence of
NDV in the inoculated egg and IHC employed to detect NDV in chicken embryo
infected at 15th i.d. was observed. Embryos infected at 15th i.d. were negative when
tested in HA-test but positive in IHC reaction (data not shown). This supports the
possibility that the virus still remains in egg membranes.
The cellular glycosylation patterns depend on the organ and age of the animal, as
revealed by the reaction with the different lectins. Organs that show a strong or very
strong reaction with Con A (α-methyl-mannopyranoside and α-D-mannose), BSA-I
(α-D-galactoside and α-linked galactose) and WGA (GalNac) show more
predisposition to be infected with NDV. Tissues with strong MAA reaction are not
always susceptible for infection with NDV. For example, almost 100% of evaluated
tracheas were positive for both NDV pathotypes when the embryos were infected at
11th i.d. This organ showed a moderate reaction with MAA. Renal tubuli presented
stronger reaction with MAA independent of the developmental stage, and were
always negative to viral presence. Hence we can conclude that the sialic acid
detected by MAA does not play a major role in the infection with NDV.
140
It is possible to observe glycosylation pattern changes after NDV infection with the
lectins employed. Further investigations (with other lectins, for example) are
necessary in order to elucidate better the carbohydrate moieties present on the cell
membrane important for a viral infection, as well as the effect of the infection on the
glycosylation pattern of the affected cells. Also, lectin/glycosylation interactions
between avian cells and other viruses may extend our knowledge on pathogenesis
and antiviral approaches.
141
VI. Abstract
Rodríguez, Julia
Lectin and immuno histochemical investigations on cellular alterations in chicken embryos following inoculation with Newcastle Disease Virus (NDV) of different virulence
Newcastle disease (ND) is a worldwide disease of poultry caused by a
Paramyxovirus type 1, genus Avulavirus. The principal receptor of this virus is 2-
deoxy-2,3-dehydro-N-acetyl-neuraminic acid.
By means of histological techniques, including histochemistry, with special emphasis
on lectin histochemistry and immunohistochemistry, tissue changes occurring in
specific pathogen free (SPF) chicken embryos infected with NDV of different
pathotypes (HB-1 and Komarov) were investigated in comparison to uninfected
chicken embryos. For this purpose, our study was divided into two main parts:
(1) Determination of the LD50 of the NDV pathotypes under study and (2) Histological
studies in chicken embryos as mentioned above to determine the distribution of free
sugar moiëties and presence of NDV in various organs of the chicken embryos.
After euthanization, the embryos were fixed in Bouin’s solution, embedded in paraffin
wax, and 5 μm sections were stained with four biotinylated lectins [Con A (sugar
specifity: α-D-mannose/glucose), BSA-1 (sugar specifity: α-D-galactose), WGA
(sugar specifity: β-D-N-acetylglucosamine) and MAA (sugar specifity: siaα-2,3-
galactose)]. Immunohistochemical staining to detect the organ distribution of NDV,
was done employing a polyclonal antibody against ND virus raised in rabbits.
Different intensities of lectin staining were observed in the uninfected control group,
indicating the presence of free sugar residues depending on the tissue type studied,
142
incubation time and the lectin used. Apical parts of the epithelial cells of esophagus,
proventriculus, trachea, lung and air sac exhibited strong to medium staining
reactions with Con A, BSA-1 and WGA. These cells showed a significantly positive
immunochemical reactions (Fisher's Exact Test <0,05) in both infected groups (NDV-
Komarov and HB-1 pathotype) at day 11th compared to day 15th of incubation, when
only 1 of the infected with NDV-Komarov pathotype and 1 infected with NDV-HB-1
pathotype were found NDV positive. Such coincidence was not observed when a
rather weak MAA reaction and distinct immuno histochemical antivirus staining were
found. The latter finding may be caused by the presence of 2-deoxy-2,6-dehydro-N-
acetyl-neuraminic acid as a second receptor of NDV not recognized by MAA. At
present, we can not explain the reaction difference in immuno histochemistry
between day 11 and day 15 of incubation, but it may be speculated that this
observation is attributable to resorption of possibly proteinaceous substances during
the process of drinking amniotic fluid, both beginning about day 13 of incubation.
Stronger lectin staining reactions were observed in infected organs of the chicken
embryos infected at 11th; when compared to the uninfected controls. In these cases,
significant differences were observed in the epithelium of the trachea with Con A,
BSA-1, and the epithelium and goblet cells in the ventriculus with MAA when they
were analyzed by Fisher´s Exact Test (�0,05). Further investigations are necessary
to elucidate the role of the different sugar moiëties in virus infection, because they
are very important structures in biological processes such as cell-cell interactions and
pathogen entry into the host cell.
In summary, the findings presented herein for the first time describe the
(1) distribution of four different sugar moieties in various organs of the chicken
embryo and (2) changes induced following infection with two different pathotypes of
NDV. This may be the basis for further pathogenesis studies employing chicken
embryos and their interaction with pathogens on the cellular level.
143
VII. Zusammenfassung
Rodríguez, Julia
Lectin- und immunohistochemische Untersuchungen der Veränderungen auf zellularer Ebene bei mit dem Newcastle-Krankheit-Virus (NKV) verschiedener Pathotypen infizierten Hühner-Embryonen
Die Newcastle Krankheit (NK) ist eine weltweit verbreitete Geflügelkrankheit, die
durch eine Infektion mit dem Paramyxovirus Typ 1, Genus Avulavirus, hervorgerufen
wird. Der bedeutendste zelluläre Rezeptor ist 2-deoxy-2,3 dehydro-N-acetyl-
Neuraminsäure.
Mittels histologischer einschließlich histochemischer und immuno-histochemischer
Techniken, die schwerpunktmäßig auf Lektinhistochemie ausgerichtet waren, wurden
Gewebsveränderungen in spezifisch pathogenfreien (SPF) Hühnerembryonen
untersucht, die mit den Newcastle Krankheit Virus (NKV) verschiedener Pathotypen
(HB-1 und Komarov) infiziert worden waren. Parallel hierzu wurden nicht infizierte
Hühnerembryos untersucht. Diese Studie wurde in zwei Teilen aufgeteilt:
(1) Bestimmung der LD50 des NDV-Pathotypen und (2) Histologische Studien an
Hühnerembryonen (s.o), um die normale Verteilung der freien Zuckerbindungen und
die Präsenz von NDV in verschiedenen Organen der Hühnerembryonen
festzustellen. Hierzu wurden mit 4 biotinylierten Lektinen gearbeitet: [Con A
(Zuckerspezifität: α-D-mannose/glucose), BSA-1 (Zuckerspezifität: α-D-galactose),
WGA (Zuckerspezifität: β-D-N-acetylglucosamine) und MAA (Zuckerspezifität: siaα-
2,3-galactose)]. Immuno- histologische Färbungen wurden mit polyklonalen
Antikörpern gegen NKV durchgeführt.
Unterschiedliche Intensitäten von Lektinfärbungen wurden in der nicht infizierten
Kontrollgruppe beobachtet, wobei die freien Zucker-Reste von der Art des Gewebes,
der Inkubationszeit und den verwendeten Lektine abhängig war. Der apikale Teil der
144
Epithelzellen des Oesophagus, Vormagens, der Trachea, Lunge und den Luftsäcken
zeigte jeweils starke bis mittlere Reaktionen mit Con A, BSA-1 und WGA. Diese
Zellen zeigten deutlich positive immunohistochemische Reaktionen (Fisher's Exact-
Test <0,05) in beiden infizierten Gruppen (NKV Komarov und NKV HB-1) am Tag 11
im Vergleich zu Tag 15 der Inkubation, wobei nur ein infizierter NKV-Komarov
Embryo und ein infizierter NDV-HB-1 Embryo gefunden wurde. Es wurde dagegen
kein Zusammenhang zwischen MAA Reaktionen und dem immunohistochemischen
Virusnachweis gefunden. Das letztgenannte Ergebnis könnte durch das
Vorhandensein einens zweiten Rezeptors (2-deoxy-2,6-dehydro-N-acetyl-
Neuraminsäure) für NKV erklärt werden, welcher nicht durch MAA nachgewiesen
wird. Zur Zeit können wir die unterschiedliche immunohistochemieschen Reaktionen
zwischen dem 11 und dem 15 Tag der Inkubation nicht erklären. Es kann aber
angenommen werden, dass diese Beobachtung mit der Resorption von
interferierenden proteinhaltigen Substanzen nach Aufnahme von Amnionflüssigkeit,
beginnend am 13. Tag der Inkubation, in Zusammenhang steht.
Eine in Vergleich zur (nicht infizierten) Kontrollgruppe signifikant positive Reaktion
von Lektinen wurde in den Organen von Hühnerembryonen beobachtet die am 11.
Tag der Inkubation infiziert worden waren. Weitere Untersuchungen sind notwendig,
um die Rolle der verschiedenen Zucker-Reste bei Virus-infektionen aufzuklären, da
es sich hierbei um wichtige Strukturen handelt, die um in biologischen Prozessen,
wie z. B. Zell-Zell-Interaktionen und dem Eintritt von Krankheitserregern in die
Wirtszelle beteiligt sind
Zusammenfassend lässt sich sagen, die vorliegenden Ergebnisse erstmalig (1) die
Verteilung der vier verschiedenen Zucker-Reste in verschiedenen Organen des
Hühnerembryos und (2) die Veränderungen welche nach Infektion mit zwei
verschiedenen Pathotypen der NKV beschreiben. Dies kann die Grundlage für
weitere Studien zur Interaktion von Krankheitserregern und verschiedenen Organen
auf zelluläre Ebene, insbesondere im Hinblick auf die Glykosylierungsmuster von
Zellen sein.
145
VIII. Resumen
Rodríguez, Julia
Investigación con lectin e immuno histoquímica sobre alteraciones celulares en embriones de pollo luego de la inoculación con el virus de la Enfermedad de Newcastle (VEN) de diferente virulencia
La enfermedad de Newcastle (EN) es una enfermedad aviar de distribución mundial,
causada por un Paramixovirus tipo 1, género Avulavirus. El principal receptor celular
para este virus es 2-deoxi-2,3-dehidro-N-acetil-ácido neuramínico.
Utilizando técnicas histológicas, incluyendo histoquímicas, especialmente lectin
histoquímica e inmuno histoquímica, se investigaron cambios en tejidos de
embriones de pollo libres de patógenos específicos (LPE) infectados con diferentes
patotipos del virus de la enfermedad de Newcastle (VEN) (HB-1 y Komarov) y se
compararon con tejidos de embriones no infectados. Para este fin, nuestro estudio
se dividió en dos partes principales: (1) Determinación de la dosis letal media (DL50)
de los patotipos a utilizar y (2) Estudio histológico en los embriones de pollo, como
fue mencionado anteriormente, con el fin de determinar la distribución normal de los
azúcares libres y la presencia del VEN en varios órganos. Posterior a la eutanasia
los embriones fueron fijados en solución de Bouin, embebidos en parafina y se
tiñeron secciones de 5 μm de grosor utilizando 4 lectinas biotiniladas [Con A (azúcar
especificidad: α-D-mannosa/glucosa), BSA-1 (α-D-galactosa), WGA (β-D-N-
acetylglucosamina) y MAA (siaα-2,3-galactosa)]. La tinción inmunohistoquímica fue
realizada utilizando un anticuerpo policlonal contra el VEN preparado en conejo.
Diferentes intensidades en la reacción a lectinas fueron observadas en el grupo
control (no infectado), indicando la presencia de residuos de azúcares libres
dependiendo del tejido observado, tiempo de incubación y lectina usada. La parte
apical de las células epiteliales del esófago, proventrículo, tráquea, pulmón y saco
146
aéreo presentaron de fuerte a mediana reacción con Con A, BSA-1 y WGA. Estas
células presentaron reacción inmunohistoquímica positiva en ambos grupos
infectados (patotipo Komarov y HB-1) a los 11 días de incubación, lo que difirió
significativamente (Fisher's Exact Test ≤0,05) de aquellas en el grupo infectado a los
15 días de incubación, en el cual sólo un embrión infectado con cada patotipo fue
observado positivo. Una coincidencia similar no fue observada para la lectina MAA,
pues se encontró reacciones débiles a esta lectina, así como reacciones variables
en inmunohistoquímica. Lo anterior puede ser consecuencia de la presencia de 2-
deoxi-2,6-dehidro-N-acetil-ácido neuramínico como segundo receptor del VEN, el
cual no es reconocido por MAA. Por el momento no podemos explicar la diferencia
en la reaccion observada en immunohistoquímica entre los 11 y 15 días de
incubación, pero posiblemente este fenómeno se deba a la reabsorción de
sustancias proteináceas durante el proceso de ingestión de líquido ammnóitico, el
cual inicia el día 13 de incubación.
Reacciones más fuertes con las lectinas se observaron en los órganos de los
embriones de pollo infectados a los 11 dias de incubación; en comparación con los
controles no infectados. En estos casos, se observaron diferencias significativas en
el epitelio de la tráquea con Con A, BS-1, y el epitelio y células glandulares del
ventrículo con MAA cuando fueron analizados con el test de Fisher (p≤0,05). Futuras
investigaciones son necesarias para dilucidar el papel de los diferentes azúcares
durante infecciones virales, ya que estas son estructuras muy importantes en
procesos biológicos como la interaccion célula-célula y de agentes patógenos con
las células hospedadoras para su infección.
En resumen, los resultados aquí presentados describen por primera vez (1) la
distribución de cuatro diferentes azúcares en diversos órganos del embrión de pollo
y (2) los cambios inducidos después de la infección con dos diferentes patotipos del
VEN. Esto puede ser la base para futuras investigaciones sobre patogénesis viral,
utilizando embriones de pollo, y su interacción con agentes patógenos a nivel
celular.
147
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170
X. Annex
Annex 1. Specific Pathogen Free (SPF) eggs
VALO, Firma Lohmann Tierzucht GmbH, Cuxhaven, Germany.
Free for: Infectious Anemia, Avian Adenovirus (Serotype 1-12), Avian
Encephalomyelitis, Lymphoid Leucosis, Virus of Avian Nephritis, Avian Reovirus,
Virus of Avianpox, Infectious bronchitis, Infectious bursal disease (Gumboro
disease), Infectious Laryngotracheitis, Influenza virus type A, Marek's disease, Avian
mycoplasmosis (M. synoviae and M. meleagridis), Newcastle disease,
Reticuloendotheliosis Virus, Salmonella pullorum and other Salmonellas and Turkey
rhinotracheitis and the corresponding specific antibodies.
Annex 2. Picric acid solution
Picric acid is diluted in distilled water until the solution be saturate.
Annex 3. Hematoxylin-Delafield formula
4 gr. hematoxilin were diluted in 25 ml alcohol absolute and 400 ml 10% ammonium
alum. Afterwards 100 ml glycerin and 100 ml methanol were added.
Annex 4. Eosin solution
Commercial eosin Y solution was diluted in 96% ethanol at 1%.
171
Annex 5. Schiff reagent
- 1 gr. Fuchsine + 400 ml distilled water + 1 ml thionyl dichloride
- 12 hours in fume hood
- 2 gr. activated carbon
- filter
- Store in refrigerator (4ºC), in amber bottle.
Annex 6. Toluidin blue
1 gr. Toluidin blue + 1 gr. Boric acid + 0,2 gr. Para formaldehyde + 100 ml distilled
water.
172
Acknowledgments
To the Deutscher Academischer Austausch Dienst (DAAD) for the partial financing of
these investigations.
Professor Neumann and Professor Mayer for their guidance on the development of
this research and for their confidence and patience to me during these years. Prof.
Dr. Martin Beer (Friedrich-Loeffler-Institute, Federal Research Institute for Animal
Health, Germany-antiserum), Prof. Dr. Schumacher (Institut für Anatomie II:
Experimentelle Morphologie, Universitätsklinikum Hamburg-Eppendorf) and Prof. Dr.
Liebler-Tenorio (Friedrich-Loeffler-Institute, Federal Research Institute for Animal
Health, Germany) for her contribution to this research
Everyone in the Poultry Clinic and in the Department of Anatomy of the Veterinary
University of Hannover, thanks for their support. Especially to Marion, Anka, Isabelle,
Christine and Silke for their friendship and technical assistance.
All my friends, especially Anna for your words of encouragement and friendship
during these years, for your help when I needed it. The whole “Tica community”,
especially to Carlos, Victor (and family), Felipe and Erika for all their support.
Jens, thanks for your support and words of encouragement every time that the
strength have left me.
To all my family for their support and affection through the distance, especially my
parents, sister and nephews.
