Advanced Laboratory Techniques in Poultry Disease Diagnosis

Dr. Joseph Giambrone
Professor
U http://www.auburn.edu/~giambjj U /
email: giambjj@auburn.edu
201 Department of Poultry Science
260 Lem Morrison Drive
Auburn University, AL 36849-5416
Teresa Dormitorio
Research Associate III
Email: tdormito@acesag.auburn.edu
Preface
ADVANCED LABORATORY TECHNIQUES IN AVIAN MEDICINE MANUAL 2

The purpose of this book is to provide the diagnostic laboratory, which is already experienced and equipped
for diagnosis of avian diseases, more advanced and sophisticated techniques for disease diagnosis. The book
is divided into two sections. The first section gives credit to the time honored traditional methods. The
second provides an introduction to newly developed techniques in molecular biology. Diagnostic methods
will be covered for infectious organisms only, which include bacteria, mycoplasma, fungi and viruses. For the
molecular diagnosis of DNA containing microorganisms, the Mycoplasma species will be used. The very
common avian viruses, infectious bursal disease and avian reoviruses, are used as an example of RNA
containing microorganisms. Only the most commonly found organisms in each group will be covered, but
the techniques are similar for less important species. The mention of any product or company name does not
imply endorsement.
Acknowledgments
This book and CD depended upon many people without whom it could not have been written. Sincere thanks
go out to former graduate student, Wayne Duck, who suggested a need for this book and thereby helped in
the preparation of some of the initial materials. We would also like to thank Loraine M. Hyde from Poultry
Science Department for her help in typing, checking, and typesetting this manuscript. Thanks to the Film
Lab of AU for there help in scanning the photos and organizing the Book.
TABLE OF CONTENTS

Page
Authors 2
Preface 3
Acknowledgments 3
TABLE OF CONTENTS 4
Introduction 5
I.TRADITIONAL DIAGNOSTIC METHODS
Isolation and Identification of Microorganisms 5
1) Bacteria
a) Salmonella 7
b) Escherichia coli 9
c) Pasteurella multocida 10
d) Staphylococcus aureus 11
e) Mycoplasma 12
2) Fungi
a) Aspergillus 14
3) Viruses 15
a) Cultivation of viruses in chicken embryos 19
Routes of inoculation and collection of specimens for avian influenza 19
b) Propagation in chicken tissues 25
c) Propagation in cell culture 26
Chicken kidney cells 27
Chicken embryo fibroblasts 28
Chicken embryo liver cells 29
Tracheal rings 30
Cell lines and Secondary cells 31
d) Application of cell culture techniques in virology 31
e) Virus Identification 32
B. Serological procedures 41
1) Immunodiffusion 45
2) Agglutination—Salmonella 47
3) Hemagglutination Inhibition—ND, MG, IBV 47
4) Immunofluorescence 49
5) Virus Neutralization—IBV, AE, IBDV 53
6) Enzyme Linked Immunoabsorbent Assay 57
C. Immunosuppression
1) Introduction 63
2) Definition 63
3) Evaluation 63
a) Antibody 63
b) CMI 64
4) Causes 64
5) Prevention 65
II. MOLECULAR BIOLOGICAL TECHNIQUES 105
A. Nucleic Acids 105
1) Propagation, purification and quantification of IBDV RNA 112
2) Quick IBDV RNA isolation procedure 129
3) Restriction fragment length polymorphism 133
a) Mycoplasma gallisepticum 136
b) Silver stain 138
4) Hybridization 142
a) Radioactive Probes 148
b) Non-radioactive Probes 150
c) Dot and Slot Blot 152
d) Southern Blot 152
e) Northern Blot 167
f) In situ Hybridization 184
g) Tissue Print Hybridization 185
h) In situ PCR 188
i) Nested PCR 192
5) Polymerase Chain Reaction 195
a) Restriction fragment length polymorphism 207
b) Real Time PCR for avian influenza 210
b) Sequencing 225
6) Microarray Assay 236
Proteins 237
1)Electrophoretic Separation 239
2)Dot and Western Immunoblots 244
3)Monoclonal antibodies—production and uses 258
a) Antigen capture ELISA 267
b) Immunoperoxidase test 271
Appendix
1.Selected list of suppliers 274
2.Procedures for Preparation of Buffers and Reagents 279
3.Commonly Used Abbreviations 285
Glossary 287
INTRODUCTION

Much of the rapid development in the poultry industry worldwide has been due to improvements in genetics,
nutrition and disease control. Knowledge of the cause of diseases has expanded dramatically over the years.
Advances in the diagnosis, treatment and vaccination have contributed to improved disease control. It is
extremely important to identify a pathogen before the disease can be adequately controlled. However, the
isolation of an organism from a lesion does not always mean that it is directly responsible for the disease.
The agent may be a secondary invader or become a primary pathogen after the bird’s immune system was
suppressed. This immunodepression could be brought about by a variety of agents and environmental
conditions. In addition, birds may be submitted late in the course of the disease and only secondary invaders
such as bacteria are readily isolated. Affected birds should be submitted as early as possible to increase the
chance of isolation of primary invaders, especially viruses.
Diseases may be caused by one or more agents. Therefore, it is important to undergo a routine battery of
tests; otherwise you may miss one or more affecting agents. It may be also necessary to collect serum from
live, diseased birds to check for abnormally high or low levels of antibody against a variety of common
infectious organisms. Submission of at least 10 birds from a disease flock is usually adequate. A combination
of normal, sick and recently dead birds and/or tissues, blood specimens, samples of feed, water and litter,
plus a thorough history of the flock should be submitted. The time honored traditional methods of isolation
and identification of disease pathogens and/or the antibodies they induce is still the backbone of the
diagnostic laboratory. However, more sophisticated techniques using molecular biological techniques such
as monoclonal antibodies, nucleic acid probes, polymerase chain reaction, and restriction fragment length
polymorphism are now being used routinely in diagnostic laboratories. It is the subject of these advanced
techniques, which sets this book apart from its predecessors.
In the chapters on molecular biology, introductory material will explain the basis of each technique after
which specific methodology will follow which gives details in step by step fashion. The specific techniques
will be centered on one DNA containing organism, e.g. mycoplasma or RNA microbe, e.g. infectious bursal
disease virus. These pathogens are featured since they are extremely important pathogens of poultry and
because much more is known about their genetic material. However, at the molecular level, genetic
manipulations are basically the same and techniques described herein can be adapted for most avian
pathogens with little modifications.
I. TRADITIONAL DIAGNOSTIC METHODS
A.Isolation and Identification of Microorganisms
Bacteria
Bacteria, along with blue-green algae, are prokaryotic cells. That is, in contrast to eukaryotic cells, they have
no nucleus; rather the genetic material is restricted to an area of the cytoplasm called the nucleoid.
Prokaryotic cells also do not have cytoplasmic compartment such as mitochondria and lysosomes that are
found in eukaryotes. However, a structure that is found in prokaryotes but not in eukaryotic animal cells is
the cell wall which allows bacteria to resist osmotic stress. These cell walls differ in complexity and bacteria
are usually divided into two major groups, the gram-positive and gram-negative organisms, which reflect
their cell wall structure. The possession of this cell wall, which is not a constituent of animal cells, gives rise
to the different antibiotic sensitivities of prokaryotic and eukaryotic cells. Prokaryotes and eukaryotes also
differ in some important metabolic pathways, particularly in their energy metabolism and many bacterial
species can adopt an anaerobic existence.
In this section, we shall look at the structure of typical bacterial cells and the ways in which they liberate
energy from complex organic molecules. Various aspects of bacterial structure and metabolism are the basis
of bacterial identification and taxonomy. Bacteria are constantly accumulating mutational changes and their
environment imposes a strong selective pressure on them. Thus, they constantly and rapidly evolve. In
addition, they exchange genetic information, usually between members of the same species but occasionally
between members of different species. We shall see how this occurs.
Bacteria have parasites, the viruses called bacteriophages which are obligate intracellular parasites that
multiply inside bacteria by making use of some or all of the host biosynthetic machinery. Eventually, these
lyse the infected bacterial cell liberating new infection phage particles. The interrelationships of bacteria and

the pages will be investigated.
Taxonomy
The basis of bacterial identification is rooted in taxonomy. Taxonomy is concerned with cataloging bacterial
species and nowadays generally uses molecular biology (genetic) approaches. It is now recognized that many
of the classical (physiology-based) schemes for differentiation of bacteria provide little insight into their
genetic relationships and in some instances are scientifically incorrect. New information has resulted in
renaming of certain bacterial species and in some instances has required totally re-organizing relationships
within and between many bacterial families. Genetic methods provide much more precise identification of
bacteria but are more difficult to perform than physiology-based methods.
Family: a group of related genera.
Genus: a group of related species.
Species: a group of related strains.
Type: sets of strain within a species (e.g. biotypes, serotypes).
Strain: one line or a single isolate of a particular species.
The most commonly used term is the species name (e.g. Streptococcus pyogenes or Streptococcus pyogenes
abbreviation S. pyogenes). There is always two parts to the species name one defining the genus in this case
"Streptococcus" and the other the species (in this case "pyogenes"). The genus name is always capitalized but
the species name is not. Both species and genus are underlined or in italics.
B. The Diagnostic Laboratory
The diagnostic laboratory uses taxonomic principles to identify bacterial species from birds. When birds are
suspected of having a bacterial infection, it is usual to isolate visible colonies of the organism in pure culture
(on agar plates) and then speciate the organism. Physiological methods for speciation of bacteria (based on
morphological and metabolic characteristics) are simple to perform, reliable and easy to learn and are the
backbone of hospital clinical microbiology laboratory. More advanced reference laboratories, or laboratories
based in larger medical schools additionally use genetic testing.
Isolation by culture and identification of bacteria from patients, aids treatment since infectious diseases
caused by different bacteria has a variety of clinical courses and consequences. Susceptibility testing of
isolates (i.e. establishing the minimal inhibitory concentration [MIC]) can help in selection of antibiotics for
therapy. Recognizing that certain species (or strains) are being isolated atypically may suggest that an
outbreak has occurred e.g. from contaminated hospital supplies or poor aseptic technique on the part of
certain personnel.
Steps in diagnostic isolation and identification of bacteria
Step 1. Samples of body fluids (e.g. blood, urine, cerebrospinal fluid) are streaked on
culture plates and isolated colonies of bacteria (which are visible to the naked eye) appear after
incubation for one - several days. It is not uncommon for cultures to contain more than one
bacterial species (mixed cultures). If they are not separated from one another, subsequent tests
can’t be readily interpreted. Each colony consists of millions of bacterial cells. Observation of these
colonies for size, texture, color, and (if grown on blood agar) hemolysis reactions, is highly
important as a first step in bacterial identification. Whether the organism requires oxygen for
growth is another important differentiating characteristic.
Step 2. Colonies are Gram stained and individual bacterial cells observed under the
microscope.
Step 3. The bacteria are speciated using these isolated colonies. This often requires an
additional 24 hr of growth.
Step 4. Antibiotic susceptibility testing is performed (optional)
The Gram Stain, a colony is dried on a slide and treated as follows: 3
Step 1. Staining with crystal violet.

Step 2. Fixation with iodine stabilizes crystal violet staining. All bacteria remain purple
or blue.
Step 3. Extraction with alcohol or other solvent. Decolorizes some bacteria (Gram
negative) and not others (Gram positive).
Step 4. Counterstaining with safranin. Gram positive bacteria are already stained with
crystal violet and remain purple. Gram negative bacteria are stained pink.
Under the microscope the appearance of bacteria are observed including: Are they Gram positive or
negative? What is the morphology (rod, coccus, spiral, pleomorphic [variable form] etc)? Do cells occur
singly or in chains, pairs etc? How large are the cells? There are other less commonly employed stains
available (e.g. for spores and capsules). Another similar colony from the primary isolation plate is then
examined for biochemical properties (e.g. will it ferment a sugar such as lactose). In some instances the
bacteria are identified (e.g. by aggregation) with commercially available antibodies recognizing defined
surface antigens. As noted above genetic tests are now widely used.
Genetic characterization of bacteria
Whole genomes of a representative strain of many of the major human pathogens have been sequenced,
and this is referred to as genomics. This huge data-base of sequences is highly useful in helping design
diagnostic tests. However, rarely are more than one or two representative genomes sequenced. There is
a lot of variability in sequences among individual strains. Thus for practical reasons, genetic comparisons
must involve multiple strains. Certain genes have been selected to define common traits among species
and then this information is used to develop diagnostic tests.
1. Sequencing of 16S ribosomal RNA molecules (16S rRNA) has become the "gold
standard" in bacterial taxonomy. The molecule is approximately sixteen hundred
nucleotides in length. The sequence of 16S rRNA differentiates bacterial species.
2. Once the sequence is known, specific genes (e.g. 16S rRNA) are detected by
amplification using the polymerase chain reaction, PCR. The amplified product is then detected
most simply by fluorescence (“real time” PCR) or by gel electrophoresis (the molecular weight of
the product).
3. DNA-DNA homology (or how well two strands of DNA from different bacteria bind
[hybridize] together) is employed to compare the genetic relatedness of bacterial strains/species. If
the DNA from two bacterial strains display a high degree of homology (i.e. they bind well) the
strains are considered to be members of the same species.
4. The guanine (G)+ cytosine (C) content usually expressed as a percentage (% GC) is
now only of historical value.
Chemical analysis
Commonly fatty acid profiling is used. The chain length of structural fatty acids present in the membranes
of bacteria is determined. Protein profiling is rapidly expanding. Characterization of secreted metabolic
products (e.g. volatile alcohols and short chain fatty acids) is also employed.
Rapid diagnosis without prior culture
Certain pathogens either can’t be isolated in the laboratory or grow extremely poorly. Successful isolation
can be slow and in some instances currently impossible. Direct detection of bacteria without culture is
possible in some cases; some examples are given below. Bacterial DNA sequences can be amplified directly
from human body fluids using PCR. For example, great success has been achieved in rapid diagnosis of
tuberculosis. A simple approach to rapid diagnosis (as an example of antigen detection) is used in many
doctor's offices for the group A streptococcus. The patient's throat is swabbed and streptococcal antigen
extracted directly from the swab (without prior bacteriological culture). The bacterial antigen is detected by
aggregation (agglutination) of antibody coated latex beads. Direct microscopic observation of certain clinical
samples for the presence of bacteria can be helpful (e.g. detection of M. tuberculosis in sputum). However,
sensitivity is poor and many false negatives occur. Serologic identification of an antibody response (in

patient's serum) to the infecting agent can only be successful several weeks after an infection has occurred.
This is commonly used in
Salmonella
Introduction
Avian salmonellosis is divisible into three diseases: pullorum disease (S. pullorum), fowl typhoid (S.
gallinarum), and paratyphoid. Pullorum and typhoid are not often seen in commercial poultry companies,
where serologic testing and eradication of positive breeder flocks is practiced, but are common in small
backyard flocks. Paratyphoid is common in commercial poultry operations worldwide. Two common
paratyphoid organisms are S. enteritidis and S. typhimurium. S. enteritis occurs in commercial layer (2% of
US) and S. typhimurium in poultry flocks. They are common causes of gastroenteritis in humans through
contaminated poultry products. In the US, S. enteritidis is not a pathogen in poultry, but is an important
cause of disease in some parts of Europe.
Salmonella are horizontally and vertically transmitted. Pullorum and paratyphoid diseases primarily affect
young poultry, whereas typhoid can occur at any age. Lesions include fibrinopurulent perihepatitis,
pericarditis, and necrosis of the intestinal and reproductive tracts.
Sample Collection
Liver, spleen, heart, gall bladder, blood, ovary, yolk sac, joints, eye and brain can be used for isolation on
non-selective media. The gut is commonly colonized by salmonellae, with the ceca most often infected.
Tissues may be ground and inoculated onto agar or broth. Gut tissues generally require selective media to
inhibit common nonpathogenic contaminants. The yolk sac of day-old chicks is a good source for isolation.
Feed, water and litter may also be taken from poultry houses. Sterile cotton swabs can be used for isolation.
Cotton swabs can be dragged along litter to check for environmental contamination or be used to check
breeder nests, laying cages or hatchery machines. Swabs can be stored under refrigeration in a sterile
holding media such as 200 gr of Bacto Skim Milk in 1 litter of distilled water.
Culture Media
None-Selective media include beef extract and beef infusion. Selective media include tetrathionate broths,
selenite enrichment broths, MacConkey's agar or eosin methylene blue agar (EMG). Selective plating media
include brilliant green (BG) agar supplemented with novobiocin (BGN) and XLD agar supplemented with
novobiocin (XLDN). Salmonella colonies on BG and BGN agar are transparent pink to deep fuchsia,
surrounded by a reddish medium. The HB2BS positive colonies on XLD or XLDN agars are jet black. Pink
colonies/to 2mm in diameter are present on MacConkey's agar and dark colonies 1mm in diameter on EMB.
A Gram stain reveals negative rods.
Rapid Salmonella Detection Techniques
A variety of rapid detection systems include enzyme immunoassay antigen capture assays, DNA probes, and
immunofluorescence. These techniques will be discussed later in the book.
Basic Identification Media
A combination of triple-sugar-iron (TSI) and lysine-iron (LI) agars are sufficient for presumptive
identification of salmonella. On TSI agar, salmonellae produce an alkaline (red) slant and acid (yellow) butt,
with gas bubbles in the agar and a blackening due to HB2BS production. Salmonellae will show lysine
decarboxylation, with a deeper purple (alkaline) slant and alkaline or neutral butt with a slight blackening
due to HB2BS production. Before doing serological screening procedures, the culture should be further
evaluated using additional identification media (Table 1.0).
Commercial kits employing more extensive tests include API—20E (Analy Lab Products, Plainview, NY), or
Enterotube I (Roche Diagnostics, Montclair, NJ) are also available.

Table 1.0. Reactions of Salmonella Cultures in Media
Media S. pullorum S. gallinarun Paratyphoid
Dextrose A A AG
Lactose - - -
Sucrose - - -
Mannitol A A AG
Maltose - A AG
Dulcitol - A AG
Malonate broth - - -
Urea broth - - -
Motility media - - +
A = Acid, G = gas produced
Figure 0.1.0 Slide agglutination
Serologic Identification
These methods including slide (figure 1.0) and plate agglutination will be discussed in a later chapter.
References
Mallison, E.T. and G.H. Snoeyenbos, 1989. "Salmonellosis." In A Laboratory Manual for the Isolation and
Identification of Avian Pathogens. Kendall/Hunt Publishing, Co. Dubuque, Iowa. pp. 3-11.
Escherichia
Introduction
Escherichia coli cause a common systemic infection in poultry known as colibacillosis. Colibacillosis
occurs as an acute septicemia, or chronic airsacculitis, polyserositis, or infectious process in young
poultry. Coligranuloma is a chronic infection resulting in lowered egg production, fertility and hatchability
in adult birds.
Clinical disease
Clinical signs are not specific and vary with bird age, duration of infection and concurrent disease
conditions. In septicaemia in young birds, signs include: anorexia, inactivity and somnolence. Lesions may

be seen as swollen, dark-colored liver and spleen and Ascities. Chronically affected birds may have
fibrinopurulent airsacculitis, pericarditis, perihepatitis, dermatitis and lymphoid depletion of the bursa and
thymus. Arthritis, osteomyelitis, salpingitis, and granulomatous enteritis, hepatitis and pneumonia may
occur in older birds.
Sample Collection
Heart, liver, lungs, spleen, bone marrow, joints and air sacs are all good specimens for isolation using a
sterile swab or needle or ground tissue. Cultures may be stored in E. coli broth upon refrigeration.
Culture media
E. coli, a gram – rod (figure 1.2), grows well in meat media, Tryptose blood, blood agar, SI medium, Lysine
iron agar (LIA), MacConkey's agar (figure 1.1). Differential biochemical media can be used such as triple iron
agar slants or identification kits (API-2OE or Enterotube I). On blue agar E. coli will show white glistening,
raised colonies 1-to-3 mm diameter and under the microscope as gram-negative rods. On MacConkey's agar
pink, 1-2 mm diameter dry colonies with dimple will be evident. On TSI slant, E. coli will produce a yellow
slant and butt with gas but no HB2BS (no black color). On SMI medium the indole reaction is positive, HB2BS
negative and motility +/-. In LIA the slant will be alkaline and the butt acid with no HB2BS production.
Figure 1.1 E. coli
Figure 1.2 Gram negative rods
References
Arp, L.H., 1989. "Colibacillosis." In a Laboratory Manual for the Isolation and Identification of Avian
Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa. pp. 12-13.
Pasteurella
Introduction

The disease caused by the infection with Pasteurella multocida, a bipolar encapsulated rod (figure 1.3),
inpoultry is called fowl cholera. It is common world wide and affects all species of birds including turkeys,
chickens, quail and wild water fowl.
Pasteurella
Figure 1.3 Bipolar encapsulated rods
Clinical disease
The disease occurs in birds of any age, but is more common in semi mature to mature birds. It can occur
as an acute septicemic disease with high morbidity and mortality, or chronic with low level of performance
in adult flocks. Signs include depression, diarrhea, respiratory signs, cyanosis, lameness and/or acute death.
Lesions include hyperemia, hemorrhages, swollen liver, focal necrotic areas in the liver and spleen and
increased pericardial fluids. Swollen joints and exudate in the wattles, comb and turbinates may be seen
in chronic cases.
Sample Collection
The organism may be isolated from the liver, spleen, gall bladder, bone marrow, heart and affected
joints with a sterile needle or swab. The organism is fairly stable on short term storage.
Preferred Culture Media
Dextrose Starch agar (DSA), blood agar or trypticase soy agar are recommended for primary isolation. On
DSA, 24 hour colonies are circular, 1-3 mm in diameter, smooth, translucent, and glistening. Colonies on
blood agar are similar to those on DSA, but appear grayish and translucent. P. multocida cells are typically
rods of 0.2-0.4 x 0.6-2.5 um occurring singular in pairs of short claims. Cells in tissues or from agar show
bipolar staining with Giemsa, Wayson's or Wright's stains. Capsules can be demonstrated by mixing a loop
full of India Ink on a slide and the colony and examining it at high magnification.
P. multocida can be further identified with biochemical tests. Fructose, galactose, glucose, and sucrose are
fermented without gas production. Indole and oxidase are produced and there is no hemolysis of blood or
growth on MacConkey's agar.
References
Rhoades, K.R., R.B. Rimler and T.S. Sandhu, 1989. "Pasteurellosis and Pseudotuberculosis." In a Laboratory
Manual for the Isolation and Identification of Avian Pathogens. Kendall/Hunt Publishing, Co.,
Dubuque, Iowa, pp. 14-21.

Staphylococcus
Introduction
Staphylococcus aureus (Figure 1.4) is frequently the cause of arthritis, synovitis, and localized abscesses
in joints, foot pads, skin, and over the breast muscle. The organism is ubiquitous in poultry houses and is
a common primary and secondary invader.
Figure 1.4 Colonies on blood agar
Clinical Disease
Staphylococcosis appears to be a classical opportunistic infection. Clinical disease is more frequent in birds
subjected to poor husbandry conditions such as overcrowding, sharp objects in the house, poor ventilation,
wet damp litter and birds that are immunosuppressed. The organism typically occurs following recent viral
or mycoplasma infections in the joints. The infection often occurs locally through a wound and then may
spread and become septicemic. The liver, spleen, and kidneys, may become swollen. S. aureus may begin as
a swelling in the breast area, foot pad or gangrenous dermatitis or as yolk sac infections from a hatchery or
breeder flock. Localized lesions may contain a white or yellow cheesy exudate. Septicemic lesions may have
necrotic and/or hemorrhage foci and cause swelling and discoloration of the tissues.
Sample Collection
Specimens for culture include blood or exudate from lesions. They can be collected from sterile swabs,
loops or by needles and syringes. No special precaution is needed for handling, transportation or storage
of materials.
Culture media
Staphylococci grow readily on ordinary media. Blood agar or thioglycollate broth supports the growth
of the organism. Selective media include Manitol-salt agar or the similar staphylococcus 110 medium.
Agent Identification
On agar cultures, staphylococci produce 1 to 3 mm diameter, circular, opaque, smooth, raised colonies in
18 to 24 hours. S. aureus are hemolytic on blood agars (figure 1.5). On manitol-salt agar, S. aureus colonies
are surrounded by a yellow halo. Colonies are examined microscopically to confirm that they contain gram-positive
cocci. A positive coagulase test will confirm they are pathogenic staphylococci. Commercially
available desiccated rabbit plasma containing either citrate or EDTA is used for the coagulase test. A
commercial coagulase test that uses microtubes (STAPHase, Analylab Products, Plainview, NY) is also
available.

Figure 1.5. Isolation of organisms from tissues
References
Jensen, M.M. and J.K. Skeeles, 1989. "Staphylococcosis." In a Laboratory manual for the Isolation and
Identification of Avian Pathogens. Kendall/Hunt Publishing Co., Dubuque, Iowa, pp. 43-44.
Mycoplasma
Introduction
Mycoplasma are tiny prokaryotic organisms characterized by their lack of a cell wall (figure 1.6). There are
numerous species of mycoplasma that infect poultry, however, the most common and pathogenic are M.
gallisepticum and M. synoviae. They are found in commercial breeder and layer flocks world wide and may
cause drops in egg production, fertility and hatchability as well as respiratory and skeletal system disease.
They may be transmitted in flocks both vertically and horizontally.
Clinical disease
M. gallisepticum (MG) is a cause of respiratory disease and egg production drops in chickens and turkeys.
Severe airsacculitis, swollen sinuses, coughing, rales, depressed weight gain, poor feed conversion, mortality
and increased condemnation in the processing plant. M. synoviae causes lesions of synovitis and respiratory
disease in chickens and turkeys.
Sample Collection
Cultures may be taken from the trachea, choanal cleft, affected joints, sinuses or air sacs, with sterile swabs.
Tissues may be shipped frozen for later isolation. Isolated culture may be shipped in broth medium by
overnight carrier.
Culture media
Mycoplasmae are fastidious organisms that require a protein based medium enriched with 10—15% serum.
Supplementation with yeast and/or glucose is helpful. M. synoviae requires nicotinamide adenine

dinucleotide (NAD), cysteine hydrochloride is added as a reducing agent for the NAD. A commonly used
media is Frey's (Table 1.1).
Figure 1.6. Mycoplasma colonies
Table 1.1. Frey's Media Formulation
Constant Amount
Mycoplasma broth base (BBL, Cockeysville, MD) 22.5 g
Glucose 3 gr
Swine Serum 120 ml
Cysteine hydrochloride 0.1 gr
NAD 0.1 gr
Phenol red 2.5 ml
Thallium acetate (10%) 2.5 to 5 ml
Penicillin G Potassium 10P6P units
Distilled HB2BO 1,000 ml
Adjust pH to 7.8 with 20% NaOH and filter sterilize
Broth cultures incubated at 37 aerobically are generally more sensitive than agar. Cultures are incubated
until the phenol red indicator changes to orange, but not yellow. This may take anywhere from 2 to 5 days.
Agar plates are examined for colonies under low magnification under regular light or with a dissecting
microscope. Colonies are usually evident after 3 to 5 days.
Tiny, smooth colonies 0.1 to 1 mm in diameter with dense, elevated centers are suggestive of mycoplasma
(Figure 1.6). Mycoplasma speciation is by serological methods using polyclonal or monoclonal antibody.
Serological tests include immunodiffusion, agglutination, enzyme linked immunosorbent assay (ELISA) and
immunofluorescence. The problem with polyclonal serum is that there can be cross reactions between MG
and MS. Also, the serum may contain antibodies against the serum present in the medium and give false
positives. Breeders given inactivated vaccines, especially vaccines against Pasteurella, may have false positive
serologic reactions up to 6 weeks post vaccination. Therefore, the use of monoclonal antibodies, to be
discussed later in this book, is most desirable.
References
Kleven, S.H. and H.W. Yoder, 1989. "Mycoplasmosis." In a Laboratory Manual for the Isolation and
Identification of Avian Pathogens, Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 57-62.

Mycology (Fungi)
Fungi are eukaryotic organisms that do not contain chlorophyll, but have cell walls, filamentous structures,
and produce spores. These organisms grow as saprophytes and decompose dead organic matter. There are
between 100,000 to 200,000 species depending on how they are classified. About 300 species are presently
known to be pathogenic for man.
There are four types of mycotic diseases:
1. Hypersensitivity - an allergic reaction to molds and spores.
2. Mycotoxicoses - poisoning of man and animals by feeds and food products
contaminated by fungi which produce toxins from the grain substrate.
3. Mycetismus- the ingestion of preformed toxin (mushroom poisoning).
4. Infection
Aspergillus
Introduction
The most common fungal disease of poultry is Aspergillosis. It is primarily a respiratory disease, but the
organism can spread to the brain and eye causing central nervous signs and blindness. The organism is
common in warm moist environments, which include hatcheries and poultry houses. Young birds are most
susceptible, since their immune system and respiratory tract cilia, responsible for trapping foreign objects,
are less developed at that age.
Clinical Disease
Aspergillus fumigatus and A. flavus are common causes of disease in commercial young poultry. The
pulmonary system is the initial point of entry, but the agent may spread to the gastrointestinal tract, eye or
central nervous system. There are two forms of the disease. The acute form occurs as brooder pneumonia in
young animals causing respiratory disease and death. The chronic form occurs in older birds and may result
in respiratory signs or torticollis and cloudy eyes. Small, white cheesy nodules may occur in acute disease in
the lungs, airsacs or intestinal tract. Plaques (yellow or gray) may occur in chronic cases in the brain or
respiratory tract.
Sample Collection
Lesions are the preferred source for culture using sterile swabs or inoculation loops. The samples may be
shipped or stored for a short time at refrigeration temperatures.
Culture Media
Initial isolation may be accomplished on blood agar, or Sabouraud's dextrose agar. Specimens can be
smeared on plates or minced in a grinder with sterile saline. The plates can be inoculated at 37C for 1 to 3
days. Chloramphenicol (0.5 g/liter) can be added to the media to inhibit bacteria growth.

Figure 2.1 Fungal culture
Small greenish blue colonies with fluffy down (Figure 2.1) can be transferred to Czapek's solution agar (Difco
Lab, Detroit, MI) for a definitive diagnosis. Scrapings of a colony or from a lesion can be placed on a
microscope slide and stained with 20% KOH. Branching septate hyphae 4 — 6 micron in diameter will be
evident. The presence of the conidial head is needed to differentiate the various species of Aspergillus.
Lactophenol, a semi-permanent mounting medium, contains 20 gr of phenol, 40 ml of glycerin, 20 ml of
lactic acid and 20 ml of distilled HB2BO. For staining hyphae and examination of conidia, 0.05 g of cotton
blue can be added to make lactophenol cotton blue. A piece of colony can be teased apart with a needle,
stained, marked and mounted with a cover slip. Species identification may be achieved on the basis of
morphological criteria upon microscopic examination.
References
Richard, J.L. and E.S. Beneke, 1989. "Mycosis and Mycotoxicosis." In a Laboratory Manual for the Isolation
and identification of Avian Pathogens. Kendall/Hunt Publishing Co., Dubuque, Iowa, pp. 70-76.
Viruses
Introduction
Viruses are important subcellular pathogens of poultry. Viruses are tiny obligate intracellular organisms.
They can only be seen with the electron microscope, and since they don't have cellular organelles or
metabolic machinery, they can only be propagated in a living host and are not affected by common
antibiotics. Important viruses of poultry include: infectious bronchitis, Newcastle disease, influenza,
laryngotracheitis, and pneumo viruses which cause respiratory diseases; Marek's disease and lymphoid
leukosis viruses, which cause lymphoid tumors, and immunosuppression; adenoviruses, chicken anemia
virus, reoviruses, and infectious bursal disease viruses, which cause morbidity, mortality and/or
immunosuppression; and fowl pox virus which causes skin and oral lesions.
Knowledge of viral replication and genetics is necessary for understanding the interaction between the
virus and the host cell. The interaction at the cellular level and progression of a particular viral infection
determines disease pathogenesis and clinical manifestations. The host immune response to the presence
of viruses will be examined later.
Interaction Between Viruses and Host Cells
The interaction between viruses and their host cells is intimately tied to the replication cycle of the virus.
Moreover, the interaction of virus with cellular components and structures during the replication process
influences how viruses cause disease. Overall, there are four possible primary effects of viral infection on a
host cell. Most infections cause no apparent cellular pathology or morphological alteration; however,
replication may cause cytopathology (cell rounding, detachment, syncytium formation, etc.), malignant
transformation, or cell lysis (death).

Cell Death
Cell death during viral replication can be caused by a variety of factors. The most likely factor is the
inhibition of basal cellular synthesis of biomolecules, such as proteins. During the replication cycle, the virus
induces the cellular machinery to manufacture largely viral products rather than those the cell would
normally make. As a result, the predominant products synthesized by the cell are viral and the cellular
products necessary for the survival of the cell are not present or present in too low a quantity to maintain its
viability. In addition to the lack of essential cellular products, this event results in accumulation of viral
products (RNA, DNA, proteins) in excess, which can be toxic for the cells. In the release phase of the
replication cycle of some viruses, apoptosis of the host cell is stimulated. In other instances, inhibition of the
synthesis of cellular macromolecules causes damage to lysosomal membranes and subsequent release of
hydrolytic enzymes resulting in cell death.
Cellular Effects
Cytopathic effect (CPE) denotes all morphologic changes in cells resulting from virus infection. Infected cells
sometimes have an altered cell membrane; as a result the infected cell membrane is capable of fusing with its
neighbor cell. It is thought this altered membrane is the result of the insertion of viral proteins during the
replication cycle. The result of fusion is the generation of a multinucleate cell or syncythia. The formation of
syncythia is characteristic for several enveloped viruses, such as herpesviruses and paramyxoviruses. The
altered cell membrane also is altered with regard to its permeability, allowing influxes of various ions, toxins,
antibiotics, etc. These multinucleate cells are large and are sometimes called "multinucleate giant cells".
Another aspect of CPE is the disruption of the cytoskeleton, leading to a "rounded up" appearance of the
infected cell. The cell in this case will either lyse or form syncythia. CPE occurrence in clinical specimens can
indicate viral infection and CPE is used as the basis for the plaque assay used in viral enumeration. Infection
of cells with some viruses (e.g., poxviruses and rabies virus) is characterized by the formation of cytoplasmic
inclusion bodies. Inclusion bodies are discrete areas containing viral proteins or viral particles. They often
have a characteristic location and appearance within an infected cell, depending upon the virus.
Malignant Transformation
In this process, viral infection results in host cells that are characterized by altered morphology, growth
control, cellular properties, and/or biochemical properties. Malignant transformation and resulting
neoplasia may occur when the viral genome (or a portion) is incorporated into the host genome or when viral
products are themselves oncogenic. Viruses causing malignant transformation are referred to as tumor
viruses. Viruses from different families have been shown to possess the ability to transform host cells. The
tumor viruses have no common property (size, shape, chemical composition) other than the development of
malignancy in the host cell. Malignant transformation is often characterized by altered cellular morphology.
This includes the loss of their characteristic shape and assumption of a rounded up, refractile appearance as
described for CPE. This is the result of the disaggregation of actin filaments and decreased surface adhesion.
Altered cell growth, the hallmark for malignant transformation, is exhibited in viral cells that have lost
contact inhibition of growth or movement, have a reduced requirement for serum growth factors, and/or no
longer respond to cell cycle signals associated with growth and maturation of the cell (immortality). Some of
the altered cellular properties exhibited by malignantly transformed cells include continual induction of
DNA synthesis, chromosomal changes, appearance of new or embryonic surface antigens, and increased
agglutination by lectins. Commonly altered biochemical properties of malignantly transformed cells include
reduced levels of cyclic AMP. Cyclic AMP is a chemical signal associated with the cell cycle and by keeping
the levels reduced the cell continually divides. Also involved is the increased secretion of plasminogen
activator (clot formation), fermentation for the production of lactic acid (known as the Warburg effect), loss
of fibronectin, and changes in the sugar components of glycoproteins and glycolipids.
Oncogenesis
Although cause-and-effect has been difficult to obtain, a number of DNA and RNA viruses have been
associated with neoplastic transformation. Viruses implicated in oncogenesis either carry a copy of a gene
associated with cell growth and proliferation or alter expression of the host cell’s copy of the gene. Effected
genes include those that stimulate and those that inhibit cell growth. Viral genes that transform infected cells

are known as oncogenes (v-onc genes), which stimulate uncontrolled cell growth and proliferation. The
discovery of oncogenes led to the finding that all cells contain analogous genes, called proto-oncogenes (c-onc
genes), which are normally quiescent in cells as they are active at some point in development. Proto-oncogenes
include cellular products such as growth factors, transcription factors, and growth factor
receptors.
DNA viruses associated with oncogenesis include the Marek’s disease virus (Herpesviridae). This virus is
typically circular episomic (independent of the host chromosome, rather than integrated) nucleic acids. The
oncogenes (v-onc) encode proteins associated with the replication cycle of the virus. RNA viruses associated
with oncogenesis include members of the family Retroviridae (e.g., avian leukosis virus). These viruses
integrate their genomes (or a copy of the genome) into the host chromosome; referred to as proviruses or
proviral DNA. Viral integration is mediated by the terminal ends of the genome, known as LTRs (long
terminal repeats). LTRs contain promoter/enhancer regions, in addition to sequences involved with
integration of the provirus into the host genome. Retroviruses can cause oncogeneses by encoding oncogenes
themselves or by altering the expression of cellular oncogenes or proto-oncogenes through insertion of their
genomes into the host chromosome close to these genes.
No Morphological or Functional Changes
In some instances, infection with viral production can occur with no discernable change in the host cell. This
is referred to as an endosymbiotic infection. This is probably dependent upon the replication needs of the
virus. Most likely the virus requires cellular processes to be active in order for viral replication to take place
and thus does not alter the features of the cell.
Pathogenesis of Viral Infections
Pathogenesis is defined as the origination and development of a disease. Viral infections can be acute,
chronic, latent or persistent. The first step in the disease process is exposure.
Exposure and Transmission
Exposure may occur by direct contact with an infected animal, by indirect contact with secretions /
excretions from an infected animal, or by mechanical or biological vectors. Transmission of virus from
mother to offspring (transplacental, perinatal, colostrum) is called vertical transmission. Transmission via
other than mother to offspring is horizontal transmission. Activation of latent, nonreplicating virus can
occur within an individual with no acquisition of the agent from an exogenous source.
Portal of Entry
Viruses enter the host through the respiratory tract (aerosolized droplets), the alimentary tract (oral-fecal
contamination), the genitourinary tract (breeding, artificial insemination), the conjunctivae (aerosolized
droplets), and through breaches of the skin (abrasions, needles, insect bites, etc.). Whether or not infection
ensues following entry depends upon the ability of the virus to encounter and initiate infection in susceptible
cells. The susceptibility of cells to a given virus depends largely on their surface receptors, which allow for
attachment and subsequent penetration of the virus.
Localized and Disseminated Infections
Following infection, the virus replicates at or near the site of viral entry (primary replication). Some viruses
remain confined to this initial site of replication and produce localized infections. An example is the common
cold and similar infections in domestic animals caused by rhinovirus. Other viruses cause disseminated
(systemic) infections by spreading to additional organs via the bloodstream, lymphatics or nerves. The initial
spread of virus to other organs by the blood stream is referred to as primary viremia. Viremia can be either
by virus free in the plasma or by virus associated with blood cells. After multiplication in these organs, there
may be a secondary viremia with spread to target organs.
The virus is transmitted in a fecal-oral fashion. It initially replicates in the cells of the tonsils, migrates to the
intestines and mesenteric lymph nodes. From the mesenteric lymph nodes, the virus enters the central
nervous system. Once in the central nervous system, the neurological symptoms of: ataxia, tremors, loss of

coordination, stiffening of the limbs, convulsions, paralysis, and coma are observed. The preference of a
particular virus for a specific tissue or cell type is known as tropism.
Mechanisms of Viral Infections
Virus replication occurs in target organs causing cell damage. The number of cells infected and/or the extent
of damage may result in tissue/organ dysfunction and in clinical manifestation of disease. The interval
between initial infection and the appearances of clinical signs is the incubation period. Incubation periods
are short in diseases in which the virus grows rapidly at the site of entry (e.g., influenza) and longer if
infections are generalized (e.g., canine distemper). Some viruses infect animals but cause no overt signs of
illness. Such infections are termed subclinical (asymptomatic or unapparent). There are numerous factors
that may influence the outcome of viral infections. These include preexisting immunity, genetics of the
animal, age of the animal, and stress related factors such as nutritional status, housing, etc.
The mechanisms by which viruses cause disease are complex. Disease may result from direct effects of the
virus on host cells, such as cell death, CPE, and malignant transformation. Alternatively, disease results from
indirect effects caused by the immunologic and physiologic responses of the host. An example of indirect
physiologic response is infection with rotavirus, which causes diarrhea in young animals and humans.
Diarrhea may be caused by rotavirus-infected erythrocytes that are stimulated to produce cytokines, exciting
enteric neurons, and inducing the secretion of excess fluids and electrolytes into the large intestine. The
virus spreads from the CNS to peripheral nerves within axons. The host responds to the presence of the
virus-infected neurons by inducing a cell-mediated immune response. Macrophages, neutrophils, and
specific cytotoxic T lymphocytes are activated to kill bornavirus-infected neurons. The result is chronic
inflammation in the CNS that corresponds with the neurological signs associated with the disease.
Two very important terms used in the discussion of microbial diseases are pathogenicity and virulence.
Pathogenicity denotes the ability of a virus or other microbial/parasitic agent to cause disease. Virulence is
the degree of pathogenicity. An avirulent virus is one lacking the capacity to cause disease. An attenuated
virus is one whose capacity to cause disease has been weakened frequently by multiple passages in cell
cultures, embryonated eggs or animals.
Virus Shedding
Virus shedding is the mechanism of excretion of the progeny virions to spread to a new host, thus
maintaining the virus in a population of hosts. Viruses are typically shed via body openings or surfaces. For
localized infections, virus is typically shed via the portal of entry. In disseminated infections, virus may be
shed by a variety of routes. Not all viruses are shed from their hosts. These include viruses that replicate in
sites such as the nervous system, as in viral encephalitis, and dead-end hosts.
Evasion of Host Defenses
In an effort to ward off the infection, the host initiates an inflammatory response. Principal components of
this response include interferons, cytotoxic T lymphocytes, antibody producing B-lymphocytes, a variety of
effector molecules, and complement. These various components work in concert and augment one another
in an attempt to rid the host of the infecting virus. In this effort to rid itself of the infecting virus, the
inflammatory response causes many of the clinical signs and lesions associated with viral infections.
Interferons (α and β) are produced by virus-infected cells. They act to stop further virus replication in the
infected and neighboring cells. Interferons also enhance antigen expression on infected cells, thereby making
them more recognizable to cytotoxic T cells. Some viruses (e.g., adenovirus) produce RNAs that block the
phosphorylation of an initiation factor, that reducing the ability of interferon block viral replication.
Cytotoxic T cells kill viral infected cells by releasing perforins, which create pores in the virus-infected cell.
Granzymes are then released into the virus-infected cell, which degrade the cell components. Lastly,
cytotoxic T cells stimulate apoptosis of the host cell.
Some viruses reduce the expression of MHC class I antigens on the surface of the host cell (e.g.,
cytomegalovirus, bovine herpesvirus type I, adenoviruses). As cytotoxic T cells cannot detect viral antigens
that are not complexed with MHC class I antigens, virus-infected cells cannot be destroyed in this manner,
allowing "survival" of the virus within the host. However, cells with no or insufficient MHC class I antigen on

their surface are recognized by natural killer cells, which kill the cell in a manner similar to that described for
cytotoxic T cells. Antibody producing B-lymphocytes secrete specific antibodies to neutralize the infectious
virions when the cell liberates them. Antigen-antibody complexes in turn can activate the complement
system. Complement aids in stimulating inflammation and the effective neutralization of virus and in the
destruction of viral infected cells.
The various effector molecules (cytokines) that are produced by the cells of the immune system have many
roles, including the induction of fever and the attraction of other inflammatory cells, (e.g., neutrophils and
macrophages) to the injured site. Some viruses possess receptors for a variety of cytokines (e.g., vaccinia
virus has receptors for interleukin-1, which stimulates fever production). When immune cells release the
cytokine, it is bound to the virus. This, in turn, reduces the amount of the cytokines available to modulate
immune responses. This enhances the "survival" of the virus within the host. An alternate mechanism to
evade the immune response is to have many antigenic types (serotypes). An immune response to one
serotype does not guarantee protection from another serotype of the same virus. For example, there are over
100 serotypes of rhinovirus and 24 serotypes of bluetongue virus.
Persistent Viral Infections
Some viruses have the ability to abrogate the inflammatory response and cause persistent infections. They
accomplish this in a number of ways, including the destruction of T lymphocytes causing
immunosuppression, the avoidance of immunologic surveillance by altering antigen expression, and by the
inhibition of interferon production.
There are three clinically important types of persistent infections:
Chronic-carrier infections
These are organisms that continually produce and shed large quantities of virus for extended periods of time.
As a result they continually spread the virus to others. Some chronic-carriers are asymptomatic or exhibit
disease with very mild symptoms. Examples include infections with equine arthritis virus, feline
panleukopenia virus, and avian polyoma virus.
Latent infections
A special type of persistent infection is one in which the virus is maintained in the host in a "non-productive"
state. Herpesviruses are notorious for causing latent infections. The viral genome is maintained in neurons
in a closed circular form, and is periodically reactivated (often during stressful conditions) resulting in a
productive infection and viral shedding. Latent infections also occur with retroviruses in which the proviral
DNA is incorporated into the host cell genome.
Cell transformation and malignancy may result if the integrated transcript causes a disruption of normal
cellular
control processes.
Slow Virus Infections
This refers to those viral infections in which there is a prolonged period between initial infection and onset of
disease. In this case, viral growth is not slow, but rather the incubation and progression of disease are
extended.
CULTIVATION OF VIRUSES IN CHICKEN EMBRYOS
Propagation of viruses is done for their initial isolation and detection, passage for stock cultures, chemical
analysis, vaccine production, preparation of antigens for serological tests, and for other immunological and
molecular needs Since viruses can only be propagated in living hosts, embryonating eggs, tissues and cell
cultures have been commonly used for their cultivation. Chicken embryos are used because of their (1)
availability, (2) economy, (3) convenient size, (4) freedom from latent infection and extraneous
contamination, and (5) lack of production of antibodies against the viral inoculum. Eggs from healthy,
disease-free flocks should be used.

Incubation of embryos is usually at 98.8—99.5F (37.1—37.5C) throughout the entire period. Lower
temperatures may be required under certain circumstances.
Knowledge of the development of the avian embryo is necessary for utilization of this medium for cultivation
of viruses. The embryo commences development as a sheet of cells overlying the upper pole of the yolk. The
embryo is recognized with difficulty during the first few days, but at 4- or 5-days of incubation it may be
readily detected by candling. From the 10th day the embryo rapidly increases in size and feathers appear. As
the embryo increases in size, there is an accompanying decrease in the volume of the extraembryonic fluids.
At the time of hatching there is no free fluid in any of the extraembryonic cavities. Throughout incubation
there is a steady loss of water by transpiration through the shell.
The amnion and chorion arise by a process of folding and overgrowth of the somatopleure. The amnion
develops first over the head and then the caudal region. By fusion of the lateral folds, the amnion completely
envelops the embryo, except for the yolk sac, from the 5th day of incubation. From the 6th to 13th days there
is an average of about 1 ml of amniotic fluid. By the 10th day, the chorion almost completely surrounds the
entire egg contents and is in immediate contact with the
shell membrane.
The allantois appears on the 3rd day as a diverticulum from the ventral wall of the hind gut into the
extraembryonic cavity and rapidly enlarges up to the 11th or 13th day. During the process of enlargement,
the outer layer of the allantois fuses with the outer layer of the amnion and the inner layer of the chorion to
form the allantoic cavity. The amount of allantoic fluid varies from about 1 ml on the 6th day to 10 ml on the
13th day. The fused chorion and allantois is known as the chorioallantoic membrane, which is highly
vascular and constitutes the respiratory organ of the embryo.
In the early stages of development, the amniotic and allantoic fluids are solutions of physiologic salts. After
about the 12th day, the protein content and viscosity of the amniotic fluid increases. The allantoic cavity
receives the output of the kidneys, and after the 12th or 13th day the allantoic fluid becomes turbid because
of the presence of urates. The yolk sac consists of a steadily enlarging sheet of cells. From the 12th day on,
the yolk material becomes progressively drier and the yolk sac more fragile. During the last 24 to 48 hours of
incubation, the yolk sac is drawn into the abdominal cavity.
Routes of Inoculation and Collection of Specimens
The various procedures outlined for inoculation of chicken embryos and for collection of specimens are a
compilation of methods. Tissues and organs from embryos and birds should be collected aseptically using
standard recovery procedures. The CAM, yolk sac and embryo or bird tissues should be ground as a 10%
suspension in a sterile diluent with antibiotics and then centrifuged at low speed (1,500 x g for 20 minutes
before inoculation).
Some of the factors influencing the growth of viruses in chicken embryos are (1) age of the embryo, (2) route
of inoculation, (3) concentration of virus and volume of inoculum, (4) temperature of incubation, and (5)
time of incubation following inoculation. The presence of maternal antibody in the yolk of hens immunized
against or recovered from certain viral infections, precludes the use of the yolk sac route for initial isolation
and subsequent passage of viruses. All fluids from live birds suspected of having virus material should have
antibiotics such as gentamicin, penicillin+streptomycin and fungizone added to it before inoculation into
embryos.
It may take several “blind” passages (no pathology), before noticeable pathologic changes take place in the
embryo, if the virus is in small amount. When working with viral infected material, one should always
practice sterile technique and work under a class II microbiological safety cabinet.
Allantoic Cavity inoculation employs embryos of 9- to 12-days incubation. The inoculum is generally 0.1—
0.2 cc. Some of the avian viruses which grow well in the allantoic entoderm are those of Newcastle disease,
infectious bronchitis, and influenza. This route has the advantage of simplicity of inoculation and collection
of specimens when large quantities of virus-infected fluid are to be obtained for use in chemical analysis,
vaccine production, and preparation of antigen for serologic tests.
1.Candle the embryos and select an area of the chorioallantoic membrane distant from the embryo
and amniotic cavity and free of large blood vessels about 3 mm below the base of the air
cell. In this area, make a pencil mark at the point for inoculation.

2.Make a similar mark at the upper extremity of the shell over the air cell.
3.Apply tincture of suitable disinfectant to the holes and allow to dry.
4.Drill a small hole through the shell at each mark but do not pierce the shell membrane.
5.Using a syringe with a 25 gauge 5/8 inch (16 mm) needle, inoculate 0.1 to 0.2 ml inoculum per
eggs by inserting the entire length of the needle vertically through the hole and injecting
the desired amount.
6.Seal the hole with glue or hot wax and return the eggs to the incubator.
7.Candle the eggs daily for 3 to 7 days for signs of death (absence of blood vessels and a dead
embryo at the bottom of the egg).
8.Eggs dying in 24 hours should be discarded, since their death was probably due to bacteria
(which can be determined by isolation of fluids in media) or trauma.
9.Embryos dying after that time should be refrigerated for 1 hour then fluids harvested and frozen
at -70C for later use.
10.Blind passage of suspected viral inoculum can be accomplished by reinoculating the allantoic
fluids every 5 to 7 days into fresh eggs until pathology or death occurs. Initial isolation of
some viruses from clinically ill birds including infectious bronchitis virus (IBV) may take
as many as 3 passages for embryo death to occur.
11.Always check live or dead embryos after harvesting fluids for evidence of pathologic changes such
as curling and stunting for IBV, and stunting and hemorrhages (Figure 3.1) for Newcastle
disease virus (NDV) or reoviruses.
Figure 3.1 Embryo pathology induced by virus in embryo on the right
12.All eggs should be disinfected before harvesting.
13.Crack the eggshell over the air cell by tapping the eggshell with the blunt end of sterile forceps.
Remove the eggshell which covers the air cell, being careful not to rupture the underlying
membranes, and discard pieces of the eggshell in disinfectant. Discard forceps in a beaker
of disinfectant.
14.Use forceps to tear the eggshell membrane, the CAM, and the amniotic membrane to release the
fluid. Depress the membranes over the yolk sac with the forceps and allow the fluid to
collect and pool above the forceps. Using a 5-ml pipette or syringe and needle, aspirate the
fluid and place it into a sterile 12 x 75-mm snap-cap tube or other suitable vial. It may be
necessary to carefully peel back the eggshell membrane from the CAM to permit a better
view of the membranes.
INCLUDEPICTURE
"http://www.fao.org/docrep/005/ac802e/ac802e08.jpg" *
MERGEFORMATINET INCLUDEPICTURE

MERGEFORMATINET
Viral isolation in embryos
15.Clarify the fluid by centrifugation at 1500 x g for 10 minutes and test the fluid for evidence of
virus infection using hemagglutination, electron microscopy, or other suitable methods.

16.Store at -70C for passage or other use.
I. Avian Influenza Isolation
Swab preparation
BHIB with Penicillin/Streptomycin (P/S)
Procedure:
Add 900 ml ddH2O to 1 L flask.
Add 37g BHI powder and dissolve completely by stirring over low heat.
Pour half of the BHIB solution into another 1L falsk and cover both with foil.
Autoclave flasks for 15 min on a liquid exhaust cycle and cool on bench top.
In hood, add 6.02g of Penicillin-G to 150 ml beaker.WEAR GLOVES AND MASK!
In hood, add 10g of streptomycin sulfate to 150 ml beaker. WEAR GLOVES AND MASK!
In hood, add 50 ml ddH2O to 150 ml beaker and dissolve P/S.
Transfer P/S/ solution to 100 ml flask and add 100 ddH2O to 100 ml.
Filter P/S solution through a 0.22um filtration device into a sterile 125mL bottle.
Label and store in refrigerator until BHIB is cool.
Once BHIB is cool, add P/S solution (50 ml to each flask) and stir.
Pour into ten sterile 125 mL bottles.
Label bottles, put tape around bottle neck/cap, and store in freezer.
To prepare sampling vials:
Using sterile technique and working under a hood pipette 1.8mL BHIB with P/S into each disposable 5mL
capped tubes.
Freeze vials in lab freezer until needed for sampling.
II. Sample collection
Collect cloacal swabs, place in tubes and store on ice. Samples can be shipped with cool packs if they will
arrive at the laboratory within 48 hours. Upon arrival in the lab, centrifuge fluid at low speed (500-1500 x g)
to sediment debris. Supernatant should be kept at 22-25 C for up to 15-60 min, to allow the antibiotics to
reduce the level of bacterial contamination. Supplementation with additional antibiotics may be needed. If
further reduction in bacterial contamination is required to reduce embryo deaths or nonviral HA activity
of egg fluids, the supernatant can be filtered through prewet 0.22-0.45-μm filter. However, filtration can
remove low levels of virus from samples and reduce isolation rates. Put samples in 3 vials/case then store
at -700C.
III. Processing cloacal swabs – inoculation
Candle 10 day old fertile eggs. Mark the edge of the air sac on viable eggs and discard dead/infertile eggs.
Arrange eggs on a plastic flat in six rows of four eggs.
Label eggs with sample/bird number and egg letter (A thru D for each sample)
Retrieve samples (Swabs in vials of BHIB) from the ultra cold freezer, thaw quickly in a 37 C water bath, and
then keep cold for the rest of the procedure, either in the refrigerator or an ice bath.Vortex samples for 1 min,
then centrifuge for 15-20 minutes at 1200 rpm.
Sanitize eggs by lightly wiping with sterile swab dipped sparingly in 5% iodine in alcohol solution.
Sanitize egg punch with 70% alcohol. Use the egg punch to puncture a small hole in the shell just above the
air sac line. Do not damage the membrane that lies just below the shell.
Inoculate eggs using a 23 gauge needle on a 1cc syringe. Pull up 0.6 ml of sample and inoculate 0.15 mL into
each egg through the punched holes, inserting the needle vertically, slightly pointed toward the front of the
egg.
Cover the holes with a small drop of Duco cement.
Incubate eggs for 48-72 hours in a 37 C humidified incubator.
Clean out and sanitize hoods with 70% alcohol.
IV. Processing cloacal swabs – harvest
Label a 96-well plate with one half-row per sample number and two sets of A-D columns.

Add 0.025mL PBS to each sample well and 0.50mL PBS to each control well.
Transfer eggs into cardboard trays and sterilize shells with a quick squirt of isopropanol.
Use an egg cracker and forceps sterilized by flaming to crack and peel away the top of the shell.
Using a sterile disposable pipette, place a drop of chorioallantoic fluid from each egg into the appropriate
well. Use a new pipet for each row/sample.
Cover tray of open eggs with foil and return to refrigerator.
After harvesting all the eggs, add 0.05mL 0.5% CRBC to each well of plate.
Cover and let sit at room temperature for 45 minutes.
Read and record results: pelleted CRBCs that run in a tear-drop shape upon tilting the plate 45 degrees
indicate a NEGATIVE well; hemagglutinated CRBCs (formed in a lattice-work, giving a solid pink
appearance to the entire well) indicate a POSITIVE well.
Harvest all the chorioallantoic fluid from each positive egg separately into a labeled tube.
Perform an HA titer on the fluid harvested from each egg.
Pool harvests from eggs of the same sample with similar titers.
Aliquot into labeled cryovials and freeze in the ultra-cold freezer.
Controls:
Cell control – Add 0.05mL PBS and 0.05 mL CRBC to the bottom row of the plate. Each well should pellet
and run in a tear-drop upon tilting. (This is a negative control that tests CRBC integrity and insures there is
no agglutinating virus present in the blood suspension).
V. Processing cloacal swabs – Inoculating Re-passes
Thaw virus samples in 37 C water bath, then keep cold for the rest of the procedure. Vortex about 1 minute.
Dilute samples in BHIB with P/S. Dilute 1:10 if re-passing to increase titer or to check a questionable (+/-)
first pass; dilute 1:100 if re-passing to make more stock.
Centrifuge diluted samples for 15-20 minutes at 1500 rpm.
Filter samples through a 0.22 um filter into a sterile labeled cryovial.
Inoculate and harvest samples as outlined above.
VI. Hemagglutination (HA) Test
Add 0.05mL PBS to each well of a 96-well plate labeled with the sample number for every two rows and “2,
4, 8, 16, 32, 64, 128, 256” (virus dilutions) across the top.
Add 0.05mL virus isolate to the first well of appropriate rows.
Mix and transfer 0.05mL across each row, discarding the final 0.05mL.
Add 0.05mL 0.5% CRBC to each well.
Cover and let sit at room temperature for 45 minutes.
Read and record results: pelleted CRBCs that run in a tear-drop shape upon tilting the plate 45 degrees
indicate a NEGATIVE well; hemagglutinated CRBCs (formed in a lattice-work, giving a solid pink
appearance to the entire well) indicate a POSITIVE well. Results are read as the inverse of the furthest
dilution producing complete agglutination, ie, the last positive well.
Controls:
Cell control – Add 0.05mL PBS and 0.05 mL CRBC to the bottom row of the plate. Each well should pellet
and run in a tear-drop upon tilting. (This is a negative control that tests CRBC integrity and insures there is
no agglutinating virus present in the blood suspension).
Chorioallantoic Membrane inoculation employs 10- to 12-day-old embryos and inoculum of 0.1-0.5 cc.
This route is effective for primary isolation and cultivation of the viruses of fowl pox, laryngotracheitis,
infectious bursal disease virus, reoviruses, which produce easily visible foci or "pocks." The chorioallantoic
membrane is a suitable site for study of the development of pathologic alterations and inclusion bodies, and
titration of viruses by the pock-counting technique (Figure 3.2).

Figure 3.0.2 Plaque on CAM
1.Candle embryos for viability.
2.Mark an area about 1/4 inch below and parallel to the base of the air cell. Disinfect with 70%
alcohol or Betadine® solutions.
3.Drill or punch a hole at this mark being very careful not to tear the shell membrane. Punch a hole
directly at the top of the air cell.
4.Holding the embryo in the same position and using a rubber bulb, draw air out
of the air cell by placing the bulb over the hole at the top of the embryo. This negative
pressure creates the artificial air cell by pulling the CAM down.
5.Using a 25-27 gauge needle, insert it into the artificial air sac about 1/8 inch and release the
inoculum. Make sure the embryo is lying horizontally for 24 hours of incubation then
return to upright position.
6.The following procedure is most commonly used for harvesting the CAM.
a) Crack the eggshell over the false air cell by tapping the eggshell with the blunt end of
sterile forceps. Remove the eggshell as close to the edge of the false air cell as
possible and discard pieces of eggshell in disinfectant. Discard the forceps in a
beaker of disinfectant.
b) Observe the CAM for signs of thickening (edema) and plaque formation.
c) Harvest the CAM by grasping it with sterile forceps, stripping away excess fluids with a
second set of forceps. Place harvested CAM in a sterile petri plate for further
examination or in a 12 x 75-mm snap-cap tube or other suitable vial for storage.
d) Freeze and store the vial containing the CAM at -70C.

Yolk Sac inoculation is performed with 5- to 8-day-old embryos and inoculum of 0.2-1.0 cc. This route can
be used for initial isolation of reoviruses.
1.Rotate the egg until blood vessels can be seen close to the margin of the air cell.These vessels may appear
as nothing more than an array of faint lines, orange in color, extending from a clear halo.
The embryo is within the area of the halo.
2.With an egg punch, make a hole in the top of the shell.
3.Use a 25-27 gauge, 1 ½-in. length needle. Insert the needle straight down into the yolk sac until its point is
one-half the depth of the egg. Aspirate some yolk material in the egg, and then reinoculate
the material with suspect virus material into the embryo.
4.For harvesting the Yolk-Sac Fluid:
a) Open the egg in the same way as described above for harvesting AAF.
b) Rupture the CAM to allow access to the yolk-sac membrane.
c) Grasp the yolk-sac membrane with forceps and carefully lift it to separate it from the embryo and
other membranes. Using a second set of forceps, strip off the excess yolk and
place the yolk sac in a sterile 12 x 75-mm snap-cap tube or other suitable vial for
storage.
d) The fluid can also be taken directly by aspiration through a large (small gauge) needle or pipette
(Figure 3.3). Store yolk sac at -70C.

Figure 3.0.3 Harvesting virus from embryos
PROPAGATION IN CHICKEN TISSUES
Many tissues of the chicken can serve to propagate viruses. One of the most common is the bursa of
Fabricius (Figure 3.4). This organ is a sac-like organ in the form of a diverticulum at the lower end of
the alimentary tract in birds. It produces B lymphocytes which can differentiate into plasma cells upon
antigen stimulation. Mature B-cell can then produce immunoglobulins, which are active against
pathogenic organisms. Infectious bursal disease virus, reoviruses, adenoviruses, lymphoid leukosis
viruses, and Marek’s disease virus will readily propagate in the bursa. Infectious bursal disease virus
(IBDV) replicates to a very high titer nearly 10P9P/ml in the bursa. This virus can cause severe morbidity,
mortality and/or immunosuppression in susceptible chickens. Specific pathogen free (SPF) chickens
between the ages of 3 to 6 weeks are commonly used for the propagation of this virus. Chickens are
normally given by eye and nose drop about 10P3P/ml of the virus. The chicks are housed in isolation
units maintained with filtered air and sacrificed 3 days post infection. The bursae are placed in NET
buffer and stored at -70C until needed. Bursae can be ground with a blender or grinder in buffer at a
10% suspension and then stored in an ultracold freezer.

Figure 3.0.4 Harvesting IBDV from inflamed bursa
References
Senne, D.A., 1989. "Virus Propagation in Embryonating Eggs." In a Laboratory Manual for the Isolation and
Identification of Avian Pathogens. Kendall/Hunt Publishing, Co., Dubuque, Iowa, pp. 176-181.
Villegas, P., 1986. "Cultivation of Viruses in Chicken Embryos." In A Laboratory Manual
of Avian Diseases. University of Georgia, Athens, GA. pp. 1-5.
PROPAGATION IN CELL CULTURE
Introduction
Cell cultures are used in laboratories for the isolation, identification, and propagation of viruses and for the
detection of neutralizing antibodies. Cell cultures have advantages over animals or embryonated eggs. Cell
cultures are economical, and they are a homogeneous population of cells, free of immunological and
hormonal influences that might affect replication of the virus. In addition, many cell lines can be stored in
liquid nitrogen and be readily available.
Aseptic technique is important even in the presence of antibiotics. Contamination can be reduced using
laminar flow hoods and using alcohol cleaned gloves and wearing a mask.
Laboratory equipment
Culture media and solutions need a large supply of pure water. The water must be deionized, double-distilled,
or both, to remove traces of cytotoxic organic and inorganic materials. Reverse osmosis followed by
glass distillation is used.
In general, cells are cultured in plastic petri dishes, flasks, or roller bottles specially coated for cell culture.
Plastic vessels are sterilized after use by autoclaving and discarded. It is possible, however, to recycle used
plastics. Other equipment that is needed include an inverted microscope, and, if cells are cultured in dishes
or tubes open to the atmosphere, an incubator in which the atmosphere can be maintained with 85% relative
humidity and 5% carbon dioxide. Incubator cleanliness is important to prevent contaminating the cultures.
Mouth pipetting is not acceptable. Use either a rubber pipette filler or an electric apparatus. An autoclave is
needed to sterilize, clean and disinfect lab ware at 20 lbs of pressure for 20 minutes.
Media and Solutions
The different cell-culture media all have the following composition:
1)A balanced salt solution.
2)A protein supplement such as serum.
3)An antibiotic mixture to control microbial contamination.
Cell culture media can be bought nearly complete (except for sera and antibiotics) commercially from many
sources in a powdered or liquid format. This medium provides the cells nutrients and conditions for growth.
Complex media such as minimum essential medium (MEM), M199 and RPMI-1640 are normally purchased

as 10x liquid concentrate or in powdered form). They are normally prepared with buffered salts such as
Hank's or Earle's. Additional vitamins, amino acids (particularly L-glutamine which is not stable at
refrigerated temperature) are added to improve growth. A typical avian cell culture growth media contains
the following:
Table 3.1. Balanced Salt Solutions (BSS) and Phosphate-Buffered Saline (PBS)
200 mM Glutamine 10ml
7.5% NaHCOB3B 29.30 ml
10x MEM 100 ml
HB2BO 760.7
Serum* (10%) 100ml
Total 1,000 ml
*Maintenance media may contain any where from 0 to 3% Fetal Bovine Serum depending on confluency and age of
the cells.
Hanks' and Earle's BSS are frequently used as bases for growth medium. Their function is to maintain a
physiological pH (7.2 to 7.6) and osmotic pressure and to provide water, glucose, and inorganic ions needed
for normal cell metabolism. They usually contain an indicator of pH, e.g., phenol red. BSS and PBS are also
frequently used to wash inocula and dead cells from cultures, to remove serum-containing media before
trypsinization, and to dilute trypsin solutions.
The stock solutions can be autoclaved, sterilized by filtration, and stored frozen or at 4C. For use, one
part of the 10x solution is added to nine parts of water, and it is finally sterilized either by filtration or
by autoclaving.
Trypsin-Versene Solution
Trypsin is needed to digest connective tissue so individual cells suspensions can be made. Stock solutions of
up to 2.5% trypsin can be prepared or obtained commercially. Sterilize by filtration. Dispense in 50-to-100-
ml quantities and freeze at -20C. Use as 1x (final concentrations: 0.05% trypsin and 0.025% versene (TV)) by
mixing 100 ml 10x TV with 900 ml of sterile glass-distilled water. It is recommended that TV be warmed to
37C before use.
Sodium Bicarbonate Solution
For pH control, sodium bicarbonate is added to the medium just before use. Various concentrations from
1.4% to 10%
have been used. Sterilize by filtration and store at room temperature.
Neutral Red Solution
A 1% solution of neutral red (Difco, Detroit, MI) can be prepared in water, sterilized by filtration and stored
at room temperature to observe pH of the medium.
Antibiotic Solution
Penicillin G sodium and dihydrostreptomycin sulfate are purchase desiccated and stored in the refrigerator
or in solution and stored frozen. A final concentration of 100 IU penicillin and 100 ug of
dihydrostreptomycin are normally used to control bacteria. Gentamicin sulfate stock solution at 10 mg/1 ml
can be substituted at a use level of 0.1 ml/10 ml of medium. To control fungi Nystatin (Mycostatin, Squibb,
New York, NY) or amphotericin B (Fungizone, Squibb) can be added at 40 IU or 2 ug per ml of medium,
respectively.
Sera
Fetal bovine (FBS) or calf serum (CS) are routinely added to media for cell cultures. The sera provide
unknown cofactors needed for cell growth. Only purchase sera, which have been tested to be free of
mycoplasma.
Sterilization
Many items are purchased sterilized. Others including neutral red, versene and water can be prepared by

autoclaving. Media containing thermolabile compounds such as amino acids, (antibiotics, serum or trypsin)
must be sterilized by filtration. Pressure filtration through membrane filters (Millipore, Corp., Bedford, MA
or Gelman Sciences, Ann Arbor, MI) is routinely used.
Preparation of primary avian cell cultures requires that organs be aseptically removed from embryos or
young chicks. Organs must be cut into small pieces and tissues dispersed into a suspension of single cells by
enzymatic digestion. They are then allowed to grow into confluence in an incubator.
Chicken Kidney Cells (CEK)
Kidney cells can be prepared from 18-20-day-old embryos or day-old to three-day-old chicks. The amounts
indicated here are for preparing kidney cells from 10-15 embryos.
1.Prepare media and trypsin solution and set in 37C water bath.
2.Spray eggs with disinfectant and allow drying under a sterile hood.
3.Using sterile technique (sterile equipment and media) remove embryos with blunt ended curved forceps
and put into tray. Wash embryos with 70% alcohol or sterile distilled water.
4.Use regular dissection methods or cut the backbone right above wing joint and separate. This exposes the
kidneys without having to touch the intestines and viscera.
5.Remove kidneys and put into glass beaker containing phosphate buffer solution (PBS) or Hank's balanced
salt solution (HBSS).
6.Pour off supernatant and clean kidneys. If there are any large chunks, mince lightly with scissors or
squeeze gently with forceps. Wash three to four times with PBS or HBSS. Use 75-100 ml PBS total.
7.Drain off the last wash and pour the tissue fragments into a trypsinization flask containing a magnetic stir
bar. Add 50-100 ml prewarmed (37C) trypsin-EDTA solution.
8.Put the flask on a stirrer base in 37C incubator and stir very slowly for 15-20 minutes.
9.When the supernatant is cloudy, shake flask, and then set it down for several minutes to let the clumps
settle out. Take out one drop of supernatant and put it on a glass slide and observe. If there are
many single cells and small clumps (two to 10 cells) with few very large clumps then it is time to
pour off the supernatant. Have ready a sterile graduated centrifuge tube with 5 ml of cold heat-inactivated
calf serum in it. (Set in a pan of ice.) Pour supernatant through gauze covered funnel
into this tube. (The serum stops the trypsin action). With fresh trypsin repeat process one to two
times (10 min. ea.) more. Do not extend trypsinization time past 1 hr. Centrifuge at 1000 RPM for
10 minutes.
10.The kidney cells (and RBC's) will pellet. Note the amount of cells obtained. Pour off trypsin solution.
Resuspend cells in 3-5 mls of minimal essential medium (MEM) or Hams F-10 with Earl's balanced
salt solution (EBSS). Add the cells to the appropriate amount of MEM (EBSS) with 10% heat-inactivated
fetal calf serum (growth media). One ml of cell pack can be resuspended in
approximately 200 ml of MEM (EBSS). Cells can be counted in a hemocytometer by resuspending
in a known amount of media. Make 1 to 10 dilutions of cells in trypan blue. You will want
approximately 2.5 x 10P6P cells/ml of media to plate out the cells. The 35 mmP2P plates require 2 ml
and 60 mm P2P plates require 5 ml. Do one plate first and observe after the cells are allowed to settle
for a few minutes.
The cells should form a monolayer in one to two days. When monolayer is formed, they may be inoculated or
if it is desirable, they may be inoculated simultaneously. After the cells have formed a monolayer, the old
media can be poured off, the monolayer washed with PBS. The cells are then ready to be infected with virus
or given maintenance media with 0-3% serum. The CEKS's are mainly epithelial cells and are used for
growth of infectious bronchitis virus, adenoviruses and reoviruses. CEFC's are mainly fibroblasts and are
used for growing Newcastle disease, infectious bursal disease, and herpes viruses.
Chicken Embryo Fibroblast (CEF)

1.Use nine-11 day old embryos. The technique described here is for three to five embryos. Spray eggs with a
disinfectant (70% alcohol is commonly used). Using sterile technique, open shell and remove
embryo with blunt ended curved forceps.
2.Place embryos in petri dish and cut off heads and limbs.
3.Transfer bodies to new petri dish or beaker containing PBS. In the beaker, the bodies can be fragmented
by carefully chopping them with sterile scissors. Another procedure that can be used when large
number of embryos is to be processed is as follows: attach a cannula to a 35 or 50 cc syringe,
remove plunger, and pour tissue chunks into barrel and force through cannula with the plunger
into a 30 ml beaker. Keep the cannula and syringe sterile and use it to draw off supernatant from
above settled tissue chunks during PBS washes.
4.Wash with PBS 3-4 times to remove red blood cells.
5.Pour tissue fragments into trypsinization flask containing magnetic stirring bar. Add about 50 ml pre-warmed
(37C) trypsin solution to flask and put on stir plate at slow speed on 37C incubator for
10-15 minutes. Stop trypsinization by adding 1ml calf serum or by placing the flask on ice for 3-5
min. Another trypsinization may be done on the clumps of tissues after the supernatant with single
cells has been decanted.
6.Strain cells through two folds of sterile gauge.
7.Centrifuge at 1000 rpm for 10 min and discard supernatant.
8.Add fresh PBS and vortex to wash and suspend cells.
9.Centrifuge again at 1000 rpm for 10 min and discard supernatant.
10.Note the amount of pelleted cells obtained. Resuspend cells in 1X MEM containing glutamine and 10%
FBS. One ml of cells can be diluted in 80 ml of media. Cells can be plated into 5, 25 or 100 ml flasks,
or in a roller bottle. Lids of containers kept in a COB2B atmosphere need to be loosened to allow
exchanges of gases. CEFC's will grow in a non- COB2B atmosphere and their lids need to be kept
tightly closed.
MEM (1X)
Glutamine 5ml 5ml
NaHCO3 14.65ml 14.64ml
H2O 375.35ml 410.35ml
FBS 50ml 50ml
Pen-Strep 10ml 10ml
NOTE: Secondary cells may be made from a confluent monolayer of CEF. Dilute trypsin solution 1:2 with
Hank's Balanced Salt Solution (HBSS). Pour off media on CEF plates, wash plates with 1 ml trypsin
solution (for 60 mm size dish) and pour off immediately. Add 2 ml trypsin solution to each plate
and incubate in 37C COB2B incubator for two to five minutes. Remove trypsinized cells from dish
with a pipette and put into centrifuge tube with 1 ml serum to stop reaction. Centrifuge 10 min. at
1000 rpm. Secondary cells may be plated 1:3 as heavy as the primary culture.
Normal CEFs CPE in CEFS

http://poisonevercure.150m.com/wi.htm
http://www.cdc.gov/ncidod/EID/vol9no9/03-0304-G1.htm
Primary Chicken Embryo Liver Cells (CELIC)
1.Use 13-15 day-old embryos and spray eggs with a disinfectant.
2.Using sterile technique, remove embryos from eggs, open embryos to expose livers.
3.Remove the livers with curved, blunt ended forceps and put them into a beaker containing sterile buffer
solution. Be sure to cut out the gall bladder before putting into the buffer.
4.Trim off any visible connective tissue or pieces of attached intestine. Mince tissue lightly with scissors or
forceps.
5.Allow the liver pieces to settle to bottom of beaker. Decant and discard buffer containing RBC's. Wash
three times or until the buffer is clear. (Usually 100 ml of buffer is enough for the collection and
washes).
6.Drain off the last wash and pour the tissue fragments into a trypsinization flask, rinsing the beaker out
with the trypsin solution. Add 50 ml prewarmed (37C) trypsin solution to the flask, which already
has a magnetic stirrer bar in it.
7.Put flask into 37C incubator and stir gently for 15-20 minutes. Check cells as for CEKC step #9.
8.Follow CEKC procedure for remaining steps.
9.Dilute liver cells 1:150 in MEM.
Chick Embryo Tracheal Rings
1.Tracheal ring cultures are organ cultures and do not form single cell monolayers. They are used for
primary culturing of many respiratory viruses. Use either embryos (19-20 day-old) or one-day-old
chickens. Open disinfected egg shell and remove embryo cutting away the yolk sac.
2.Cut skin until trachea is completely exposed.
3.Carefully remove the trachea with forceps and remove all fatty tissue surrounding it.
4.Place trachea in glass petri dish containing approximately 5 mls of HBSS.

5.Lay tracheas on sterile filter paper and place on tissue chopper. Use sterile razor blade and cut trachea into
rings at medium speed.
6.Wash mucous from inside of rings with a syringe and needle containing HBSS. Place rings in a separate
petri dish containing HBSS.
7.With small forceps, place individual rings into sterile test tubes. Cover with 0.5-1.0 mls of media. Be sure
rings are immersed in solution.
8.Put tubes in rack and rotate at 37C for 24 hours. Mucous may again need to be washed from inside of
the rings with HBSS.
9.At the end of 24 hours, check for ciliary movement under the microscope (use either the 4X or 10 X
objectives).
10. Score the ciliary movement as follows:
If half the ring has movement, the ring would be assigned a 2.
If 3/4 of the ring has movement, the ring would be assigned a 3.
If the entire ring has movement, the ring would be assigned a 4.
Rings with reading lower than 2 are not used.
11.The rings are now ready to be inoculated. The ciliary movement should be read after 3-5 or 7 days,
depending upon the virus being studied.
Tracheal rings can be used to detect the presence of infectious bronchitis virus (IBV), Newcastle disease
virus (NDV), and laryngotracheitis virus (LT). They can also be used to run Virus Neutralization Tests for
IBV. Tracheal rings can also be used to evaluate ciliary activity after challenge with field isolates. Rings are
prepared from adult birds four days after challenge. The ciliary activity is evaluated as described.
Procedure for Inoculating Preformed Monolayers
1.Swirl plate to resuspend as many RBC's and debris as possible and then decant and discard growth
medium.
2.Wash monolayer gently with 2-3 mls of prewarmed PBS and discard.
3.Add 0.1 ml sample inoculum to the small 10 x 35 mm plates or 0.2 ml for the larger size (60 mm). Rock
each plate gently to distribute inoculum evenly over the cell monolayer.
4.Incubate inoculated cultures in 37C incubator for 45 minutes to allow virus to absorb Rock tray once or
twice during incubation if possible.
5.Add 2 ml maintenance medium to each 35 mm plate (or 5 ml for 60 mm plates.)
Note: Maintenance media — 0% - 3% heat-inactivated calf serum.
6.Incubate at 37C. Check plates daily for damage to the cells or cytopathogenic effect (CPE).
7.To harvest samples, freeze plates and then thaw two to three times, shaking flask when media is partially
thawed to help dislodge cells and collect. Alternatively, cell monolayers can be removed by
removing media and then scraping adherent cells with a sterilized "rubber policeman". Virus will be
present both extracellularly in media and intracellular in cells. Freezing and thawing or sonication
for five minutes will disrupt cells to remove virus. For some highly intracellular viruses such as
herpes viruses it is best not to disrupt the cells.
Cell Lines and Secondary Cultures
A cell line is a population of cells derived from an animal tissue, which can be continually propagated over
numerous passage mammalian cell lines which support avian virus growth. They include VERO and BGM—
70 cells. VERO cells are derived from kidney tumors of African green monkeys whereas BGM cells are kidney
cells derived from a bovine tumor. The tumor cells’ genetic material allows them to grow indefinitely. Avian
viruses such as Newcastle disease, infectious bronchitis, infectious bursal disease and reoviruses have been
adapted to these cell lines. Cell lines from tumors of ducks and chickens will also support avian

herpesviruses, avian leukosis viruses, Marek's disease virus and the chicken anemia virus. Primary Chicken
Embryo Fibroblast Cells (CEFC's) that can be passaged up to four times in cell culture are called secondary
cells. The advantages of all lines and secondary cells are that they don't require live animals embryos and can
be stored frozen in liquid nitrogen so they are readily available when needed. The passage and use of these
lines or cells is as described under CEFC's.
Freezing cells
A low temperature freezer (-70C) or liquid nitrogen container (-196C) is needed. The freezer should be
plugged into an electric surge protector and be equipped with an alarm in case of temperature rise, and
backed up with a gas powered electric generator in case of long term power failure. The liquid nitrogen tank
should be checked monthly with a ruler to measure depth of nitrogen in the tank. The tank normally needs
to be refilled every four to six weeks depending on usage.
Procedure for freezing cells
1)Use only actively growing cells (2 to 5 days of age).
2)Prepare the cells as outlined for passage of secondary CEFC's.
3)Centrifuge the cells at 400 g for 5 mins and discard the supernatant fluid.
4)Resuspend cells in cold culture medium or calf serum containing 10% dimethyl sulfoxide.
5)Transfer the cells to prechilled freezing vials and place in an insulation container which allows for a
gradual drop in temperature of 1C per minute. Place the container in a -20C freezer for 1 hr.
then -70C for 8 hours and, if available, liquid nitrogen. Cells are viable for months at -70C and
for years at -196C.
6)For use, cells should be thawed in a water bath at room temperature.
7)Thawed cells should be plated at 2x the density of primary cell lines. Maintenance of these cells is as
previously mentioned.
Application of Cell Culture Methods for Virology
Virus multiplication in cell culture can be detected in several ways:
1)Morphologic alternation of the cell, called cytopathic effect (CPE) due to degeneration of cellular
organelles. The CPE can be seen as holes in the monolayer (Figure 3.4).

Figure3.4 Viral CPE (hole in monolayer)
Prior to death, cells may round up, become refractile or partially detach from the monolayer.
2)The formation of giant cells or syncythia (fusion of cell membranes).
3)The pH changes in the medium (red to yellow color change) due to changes in cell metabolism.
4)Serologic methods such as fluorescence or immunoperoxidase assays, can detect viral multiplication in
cells. As with chicken embryos, viruses upon initial isolation may have to be passaged blindly (no
visible CPE) several times before their presence becomes apparent.
VIRUS IDENTIFICATION
Animal viruses are classified based on their physical and chemical characteristics. Viruses are first divided
into two groups based on their nucleic acid content. Deoxyribonucleic acid (DNA) viruses are divided into
seven families, five of which contain avian pathogens. These families also contain either single or double
stranded nucleic acid. Ribonucleic acid (RNA) viruses are divided into 16 families, nine of which cause
disease in poultry. Some of the DNA families and representative viruses are 1) Adenovirus — inclusion body
hepatitis; 2) Herpes virus — Marek's disease, and 3) Pox virus — Fowl pox. Some of the most important RNA
virus families and representative individuals include: 1) orthomyxovirus — Avian influenza; 2)
paramyxovirus — Newcastle disease; 3) coronavirus — infectious bronchitis; 4) Retrovirus — leukosis; 5)
picorna virus — avian encepholamyelitis; 6) reovirus — viral arthritis; and 7) birna virus — infectious bursal
disease. Reoviruses and birna viruses contain double stranded RNA.
Other Criteria for classifying viruses include:
1)Presence of a lipoprotein envelope; 2) diameter of the virion, and 3) symmetry of nucleocapsid.
Knowledge of these criteria will help place the virus in a recognized family, however, to positively identify a
virus serologic methods (reacting an unknown virus with a known antibody) are often required (Table 3.2).
Table 3.2. Important Biological, Physico-chemical Properties of Enveloped and
Nonenveloped Virions
Characteristic Nonenveloped Virus Enveloped Virus
Ultraviolet radiation Sensitive Sensitive
Gamma radiation Sensitive Sensitive
Thermostability Thermostable Thermolabile
Susceptibility to ice crystal damage Yes Extensive
Inactivation by lipid solvents
No Yes
and detergents
Determining type of nucleic acid
The type of nucleic acid (either DNA or RNA, but not both for viruses) can be determined by various specific
inhibitors that affect virus replication. Thymidine analogs are a simple method to determine if the virus
contains DNA. 5'-iodo-2'- deoxy uridine (IUDR) (Calbiochem Corp, San Diego, CA) is commonly used. A

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