Advanced Lab Techniques in Avian Medicine

ADVANCED LABORATORY TECHNIQUES
in
AVIAN MEDICINE
Dr. Joseph J. Giambrone
Professor
and
Teresa Dormitorio
Research Associate III
201 Department of Poultry Science
260 Lem Morrison Drive
Alabama Agricultural Experiment Station
Auburn University, AL 36849-5416
Revised 7/13/2013

2
Dr. Joseph Giambrone
Uhttp://www.auburn.edu/~giambjjU/
email: giambjj@auburn.edu
Teresa Dormitorio
Email: tdormito@acesag.auburn.edu

Preface
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 and to
Kejun Guo for his help in editing and transfer of the document from MS word to adobe.
3

4
TABLE OF CONTENTS
UPage
Authors 2
Preface 3
Acknowledgments 3
Table of Contents 4
Introduction 6
I. TRADITIONAL DIAGNOSTIC METHODS
A. Isolation and Identification of Microorganisms 7
1) Bacteria
a) Salmonella 10
b) Escherichia coli 13
c) Pasteurella multocida 15
d) Staphylococcus aureus 17
e) Mycoplasma 19
2) Fungi
a) Aspergillus 21
3) Viruses 23
a) Cultivation of viruses in chicken embryos 29
Routes of inoculation and collection 30
of specimens for avian influenza
b) Propagation in chicken tissues 39
c) Propagation in cell culture 40
Chicken kidney cells 42
Chicken embryo fibroblasts 43
Chicken embryo liver cells 45
Tracheal rings 46
Cell lines and Secondary cells 47
d) Application of cell culture techniques 49
in virology
e) Virus Identification 50
B. Serological procedures 64
1) Immunodiffusion 71
2) Agglutination—Salmonella 73
3) Hemagglutination Inhibition—ND, MG, IBV 74
4) Immunofluorescence 78
5) Virus Neutralization—IBV, AE, IBDV 84
6) Enzyme Linked Immunoabsorbent Assay 91

5
C. Immunosuppression
1) Introduction 100
2) Definition 100
3) Evaluation 101
a) Antibody 102
b) CMI 102
4) Causes 102
5) Prevention 102
II. MOLECULAR BIOLOGICAL TECHNIQUES 104
A. Nucleic Acids 105
1) Propagation, purification and quantification 112
of IBDV RNA
2) Rapid IBDV RNA isolation procedure 130
3) Restriction fragment length polymorphism 134
a) Mycoplasma gallisepticum 137
b) Silver stain 141
4) Hybridization 142
a) Radioactive Probes 147
b) Non-radioactive Probes 149
c) Dot and Slot Blot 161
d) Southern Blot 166
e) Northern Blot 173
f) In situ Hybridization 183
g) Tissue Print Hybridization 184
h) In situ PCR 187
i) Nested PCR 189
5) Polymerase Chain Reaction 194
a) Restriction fragment length polymorphism 205
b) Real Time PCR for avian influenza 211
c)AIV MolecularTechniques 223
D ILTV detection 261
e) Loop Mediated Lamp 275
e) Taqman for reoviruses 294
f) Syber Green PCR 311
g) Sequencing 324
6) Microarray Assay 338
B. Proteins 339
1) Electrophoretic Separation 342
2) Dot and Western Immunoblots 345
3) Monoclonal antibodies—productionand uses 357
a) Antigen capture ELISA 365
b) Immunoperoxidase test 370
Appendix
1. Selected list of suppliers 373
2. Major sites for molecular biology on the world wide web 378
3. Procedures for Preparation of Buffers and Reagents 378
4. Commonly Used Abbreviations 384

7
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.
UTABLE of CONTENTS

8
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

9
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

10
(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

11
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 HB
2BS 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.

12
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 HB
2BS production. Salmonellae will show
lysine decarboxylation, with a deeper purple (alkaline) slant and alkaline or neutral butt with a slight
blackening due to HB
2BS 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 1.0. Slide agglutination

13
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.
Table of Contents

14
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

15
Figure 1.1. E. coli
butt with gas but no HB
2BS (no black color). On SMI medium the indole reaction is positive, HB
2BS
negative and motility +/-. In LIA the slant will be alkaline and the butt acid with no HB
2BS production.
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.
UTable of Contents

16
PASTEURELLA
Introduction
The disease caused by the infection with Pasteurella multocida, a bipolar encapsulated rod
(figure 1.3), in poultry is called fowl cholera. It is common world wide and affects all species of birds
including turkeys, chickens, quail and wild water fowl.
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.
Pasteurella
Figure 1.3. Bipolar encapsulated rods

17
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.
UTable of ContentsU

18
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.
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.

19
Figure 1.4. Colonies on blood agar
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.
UTable of ContentsU

20
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

21
P
P
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 106
units
Distilled HB
2BO 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.
Agent identification
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.
UTable of ContentsU

22
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.

23
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
Agent Identification
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 HB
2BO. 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.
UTable of Contents

24
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

25
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

26
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

27
(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.

28
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.

29
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.

30
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.

31
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).

32
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.
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.
Figure 3.1. Embryo pathology induced by virus in embryo on the right

33
Figure 3.2. Plaque on CAM
Figure 3.3 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.

34
I. Avian Influenza
Figure 3.4 Avian virus morphology
AIV is in a family of negative-sense, single-stranded RNA viruses. They are smaller than the
paramyxoviruses and their genome is segmented (7 to 8 segments) rather than consisting of a
single piece of RNA. Influenza viruses are the only members of Orthomyxoviridae.
Viruses of this family have a predilection for the respiratory tract, but usually do not cause a serious
disease in uncomplicated cases. Exceptions are human infections with viruses of avian origin.
Principal viruses of veterinary importance are type A influenza viruses, which cause equine, swine,
and avian influenza.
Viral Characteristics
• Viruses have a segmented single-stranded RNA genome, helical nucleocapsids (each RNA
segment + proteins form a nucleocapsid) and an outer lipoprotein envelope. The segmented
genome facilitates genetic reassortment, which accounts for antigenic shifts. Point mutations in
the RNA genome accounts for antigenic drifts that are often associated with epidemics. In either
case, the changes are frequently associated with the HA (hemmaglutinin) and NA
(neuraminidase) antigens.
• The envelope is covered with two different kinds of spikes, a hemagglutinin (HA antigen) and a
neuraminidase (NA antigen). In contrast, the hemagglutinin and neuraminidase activities of
paramyxoviruses are in the same protein spike.
• In the laboratory, the virus replicates best in the epithelial cells lining the allantoic cavity of
chicken embryos.
• The viruses agglutinate red blood cells of a variety of species.
• Replication takes place in the nucleus.
• The viral RNA-dependent RNA polymerase transcribes the negative-sense genome into
mRNA.
• Influenza viruses are labile and do not survive long on premises.
is in a family of negative-sense, single-stranded RNA viruses. They are smaller than the
paramyxoviruses and their genome is segmented (7 to 8 segments) rather than consisting of a
single piece of RNA. Influenza viruses are the only members of Orthomyxoviridae.
Viruses of this family have a predilection for the respiratory tract, but usually do not cause a

35
serious disease in uncomplicated cases. Exceptions are human infections with viruses of avian
origin. Principal viruses of veterinary importance are type A influenza viruses, which cause equine,
swine, and avian influenza.
Viral Characteristics
• Viruses have a segmented single-stranded RNA genome, helical nucleocapsids (each RNA
segment + proteins form a nucleocapsid) and an outer lipoprotein envelope.
• The segmented genome facilitates genetic reassortment, which accounts for antigenic shifts.
Point mutations in the RNA genome accounts for antigenic drifts that are often associated with
epidemics. In either case, the changes are frequently associated with the HA (hemmaglutinin)
and NA (neuraminidase) antigens.
• The envelope is covered with two different kinds of spikes, a hemagglutinin (HA antigen) and a
neuraminidase (NA antigen). In contrast, the hemagglutinin and neuraminidase activities of
paramyxoviruses are in the same protein spike.
• In the laboratory, the virus replicates best in the epithelial cells lining the allantoic cavity of
chicken embryos.
• The viruses agglutinate red blood cells of a variety of species.
• Replication takes place in the nucleus.
• The viral RNA-dependent RNA polymerase transcribes the negative-sense genome into
mRNA.
• Influenza viruses are labile and do not survive long on premises.
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.

36
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 -700
C.
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.

37
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) (figure 3.5).

38
Figure 3.5. HA test results
-HA test (top wells) and +HA test (bottom wells)
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).
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.

39
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.
Figure 3.6 Yolk sac injection
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 (figure 3.6).
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.

40
P
P
P
P
d) The fluid can also be taken directly by aspiration through a large (small gauge) needle
or pipette (Figure 3.7). Store yolk sac at -70C.
Figure 3.7. 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. 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 109
/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 103
/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.
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.
UTable of ContentsU

41
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

42
(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% NaHCOB
3B 29.30 ml
10x MEM 100 ml
HB
2BO 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.

43
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).

44
P P
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 106
cells/ml of media to plate out the cells.
2 2
The 35 mmP
P
plates require 2 ml and 60 mm P
P
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.

45
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 COB
2B atmosphere need to be
loosened to allow exchanges of gases. CEFC's will grow in a non- COB
2B 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 COB
2B 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

46
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.

47
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

48
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.

49
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.

50
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 3.3).
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 (figure
3.8).
Figure 3.8 Viral CPE (hole in monolayer)
UTable of ContentsU

51
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
Gamma radiation
Thermostability
Susceptibility to ice crystal damage
Sensitive
Sensitive
Thermostable
Yes
Sensitive
Sensitive
Thermolabile
Extensive
Inactivation by lipid solvents
and detergents
No Yes
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 simple method is as follows.
1. Prepare maintenance medium with 50 ug/ml IUDR. The media must be homogenized and
sterilized by filtration (Run sterility check on each concentration). Prepare dilutions of virus
with and without IUDR.

Advanced Lab Techniques in Avian Medicine

Advanced Lab Techniques in Avian Medicine

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Viewers also liked

Viewers also liked (6)

Similar to Advanced Lab Techniques in Avian Medicine

Similar to Advanced Lab Techniques in Avian Medicine (20)

More from Joseph Giambrone

More from Joseph Giambrone (9)

Recently uploaded

Recently uploaded (20)

Advanced Lab Techniques in Avian Medicine