a_-

Agriculture Handbook No. 404

NOV 16 1973

PROCUREMENT ^¿fN

CURRENT StRlAL BtCwi^a

PROCEDURES FOR THE IN VITRO ASSAY

OF VIRUSES AND ANTIBODY

OF AVIAN LYMPHOID LEUKOSIS

AND MAREK'S DISEASE

Agricultural Research Service

UNITED STATES DEPARTMENT OF AGRICULTURE
Foreword

The methods outlined herein were originally compiled from instructions

prepared in 1962 at the Regional Poultry Research Laboratory (RPRL),

East Lansing, Mich. In 1964 these instructions were revised and expanded.

However, development of newer techniques and information on lymphoid

leukosis and the rapid progress in the study of Marek's disease soon outdated the 1964 edition; hence, the present second revision is necessary.

The methods include those of many investigators in avian leukosis and cell

culture as well as simplified and improved techniques developed at the

RPRL.

Trade names are used in this publication solely

for the purpose of providing specific information.

Mention of a trade name does not constitute a

guarantee or warranty of the product by the U. S.

Department of Agriculture or an endorsement by

the Department over other products not mentioned.

Contents

Page

Introduction 1

Cell-culture equipment and techniques . 2

Equipment 2

Media preparation—stock solutions 3

Working media preparation and solutions— 4

Cell preparations - --- 5

Virus preparations ^^— 7

Rous sarcoma virus (RSV) 7

Lymphoid leukosis virus (LLV) .. 8

Marek's disease (MD) 8

Virus assay 9

Lymphoid leukosis - .^. 9

Marek's disease . 14

Antibody assay 15

Lymphoid leukosis 15

Marek's disease 15

Literature cited _.-_ , 16

Appendix 18

Sources of equipment and media 18

Use of the hemacytometer „ 18

Washington, D. c. Issued August 1971

For sale by the Superintendent of Documents, U.S. Government Printing Office

Washington, D.C. 20402-Price 20 cents
PROCEDURES FOR THE IN VITRO ASSAY OF VIRUSES

AND ANTIBODY OF AVIAN LYMPHOID LEUKOSIS

AND MAKER'S DISEASE

By J. J. SOLOMON, microbiologist, P. A. LONG, research veterinarian, and W. OKAZAKI, micrebiologistf

Aniinal Science Research Division, Agricultural Research Service

INTRODUCTION

Avian leukosis remains one of the most important and costly problems in the poultry industry today ; and, although much progress has

been made in understanding the disease or

diseases, effective control or prevention is still

not available. Losses in the United States alone

amount to well over $150 million annually, and

the disease is rampant in other countries. Therefore, effective methods for studying the causative agent or agents and immunological responses are vital, not only because of the poultry

industry but also because of possible applications in other areas of cancer research. In vitro

techniques have rapidly become of the utmost

importance in the study of the avian leukosis

complex, particularly since the advent of the

RIF test in 1960.

The RIP test was first described by Rubin

(33)^ who demonstrated the presence of a transmissible agent in chick embryo fibroblasts

(CEF) and in the growth medium of some CEF

cultures that would induce resistance to neoplastic transformation of cells superinfected

with Rons sarcoma virus (RSV). This agent

was named RIF, an acronym for ''resistance

inducing factor," Subsequent investigation

showed that RIF activty was characteristic of

many viruses of the avian leukosis complex^

{13, 33, 3If, 37, U)^ Soon afterward this characteristic became one of the important criteria

for classification of diseases in this complex.

^ItaUc numbers in parentheses refer to Literature

Cited, p 16.

- BuRMESTER, B. R., FREDRICKSON; T. N., and SOLOMON, J. J. Unpublished data. 1963.

These are currently classified into two groups,

lymphoid leukosis (LL) for lymphoid proliferations caused by RIF positive viruses and

Marek's disease (MD) for lesions of the nervous

system caused by RIF negative agents {6),

The RIF test was followed in a short time by

several other important developments, most of

which will be mentioned only briefly. Perhaps

the most significant was the discovery of a Rous

associated virus (RAV) in 1962(^4), since this

paved the way for many subsequent important

contributions. These include the discovery in

1965 by Vogt and Ishizaki {Al, h2) and Hanafusa {15) that the RIF positive viruses occur

in antigenically distinct subgroups, currently

believed to be four {11) \ the finding that susceptibility to LL infection is determined by

single autosomal genes specific for each subgroup {10) \ the observations on the defectiveness of RSV {16) and the production of nonRSV-producing cells (NP cells) {16, 39) ; and

the role of helper lymphoid leukosis viruses in

the maturation of RSV {16, 17, 40).

The observation that tumors could be induced in hamsters by RSV {1, 18) was also an

important contribution for in vitro analysis of

lymphoid leukosis virus (LLV), since it led to

the development of a complement fixation test

for avian leukosis {35) known by its acronmyn

COFAL. This procedure was found to be as

effective as the RIF test^ {14, 29) and less

stringent in cultural requirements.

^ SOLOMON, J. J., and OKAZAKI, W. Unpublished data.

1966.
Early in the 1960's Marek's disease was recognized as a major cause of leukosis mortality

or condemnation in the poultry industry ; hence,

research emphasis rapidly shifted to this

disease. These efforts culminated a few years

later in the simultaneous development in two

widely separated laboratories of cell culture

assays a^nd the identification of the agent as a

member of the herpes virus group (.9, 2Jf, 88),

As a result of these and subsequent findings,

prognosis for the control of MD appears better

now than at any previous time. It is hoped that

this publication on in vitro methods used at the

Regional Poultry Research Laboratory, U. S.

Department of Agriculture, and elsewhere will

assist in the continued research on the avian

leukosis complex and aid in its eventual control.

It is not meant to serve as a comprehensive

review or an all-inclusive publication on techniques.

CELL-CULTURE EQUIPMENT AND TECHNIQUES

(The superscripts (^-") refer to sources

for the equipment or media in the Appendix,

p. 18,)

Equipment

Reusable Glassware

Glassware normally found in microbiology or

tissue-culture laboratories are needed. Highuse items include screwcap erlenmeyer flasks,

large-volume (25- and 50-mL) serological pipettes, filtration flasks with bottom takeoff outlets, aseptic filling bells, large aspirator bottles,

and graduated cylindrical bottles.^

Disposable Glass and Plastic

Tissue-culture plastic petri dishes*' of three

sizes are used routinely. The 150- by 25-mm.

size is used primarily for growth of primary

cells, the 100- by 20-mm. size for passage of

cells, and the 60- by 15-mm. size for assay of

virus. Smaller sizes are also available and are

occasionally used for special purposes. The

special tissue culture dishes must be used. Disposable glass prescription bottles,"* 16- and 32-

ounce, are used with or without washing for

media storage. Disposable glass^ and plastic**

pipettes and syringes'^ are also available and

extensively used. The cost of the disposable

items is probably less than the cost of washing

and replacing expensive reusable glassware.

Vacuum-formed plastic containers ^ are useful

as substitutes for glass beakers and for many

miscellaneous uses.

Incubators

COo incubators ^ are used when cells are grown

in petri dishes. A concentration of 3 to 5 percent CO. in air is desirable for most studies,

and this can be obtained with now meters. An

alternate procedure is to adjust the C0:> concentration until a pH of 7.0 to 7.4 is obtained in

sterile m.edia placed in the incubator. Too little

COo gives poor growth; whereas, too high a

concentration is detrimental to the cells and

may prevent the formation of RSV foci in infected cells. CO2 is obtained commercially in

cylinders and is mixed with air supplied by a

small compressor ^ equipped with a storage

tank. The incubators may be used dry, but a

high humidity is preferred to reduce the evaporation of nutrient fluid. This may be attained

by adding water to the interior chamber of the

incubator. Water-jacketed incubators maintain

the temperature inside the incubator with less

fluctuation and variation.

Microscopes

An inverted microscope*' is used to count

RSV foci and MD microplaques in petri dishes

and for general observation of cell monolayers.

Magnifications most frequently used are between 25 and 50 X- A 25 X magnification is

used for enumerating foci or microplaques,

and the 50 X is used for study of finer cefl

details. For these magnifications the most useful combinations are a 10 X ocular with a 2.5 or

5.0X objective. A regular laboratory light

microscope is used for counting cells in the

hemacytometer.
Miscellaneous

Other equipment usually found in microbiology laboratories, such as an autoclave,

sterilizing oven, stirrers, mixers, and hoods for

aseptic procedures, is necessary. The hoods can

be fabricated locally. The vortex type mixers

are especially useful for dispersing cells.

Media Preparation—Stock Solutions

Medium 199 J—^10 X, Morgans, with L-glutamine, without NaHCOa.

Nutrient mixture FlOJ—10 X y Ham's, with Lglutamine, without NaHCOa.

BME basal mediumJ—10 X, Eagle, with Lglutamine, without NaHCO^.

Tryptose phosphate broth (TPB), dehydrated

potvderJ—The medium is rehydrated by dissolving 29.5 g. in 1,000 ml. water, and then

dispensed in aliquots in 250-ml. erlenmeyer

flasks or screwcap botl^les. Then the flasks are

sterilized in the autoclave for 15 minutes at

15 p.s.i.

Calf serum.—Purchased commercially.^ ^' Calf

serum usually does not contain avian leukosis

viral inhibitory substances when used at levels

less than 10 percent. However, routine screening of calf serum before use is recommended,

and all batches are tested for anti-leukosissarcoma virus activity and cell toxicity factors.

Fetal calf serum is known to be inhibitory to

RVS focus formation as is a high concentration

(over 10 percent) of ordinary calf serum (32).

Fetal serum has also been found to inhibit MD

induced CPE in duck embryo cells. Newborn

calf has been found to be satisfactory.

Calf serum received unfrozen is immediately

inactivated at 56" C. for 30 minutes, then dispensed in about 100-ml. quantities, and refrozen

until used. Serum received frozen is not thawed

until used, at which time it is also inactivated

as above.

Chicken serum,—Chicken serum when used

as a nutrient or growth factor in cell culture

should be free of LLV and RSV antibody, although there is some evidence that antibody

may only mask virus without eliminating viral

production by the cells (3). Most of the commercial chicken sera have been found to contain

RSV antibody. Thus, RSV antibody-free serum

must be obtained from uninfected stock, and the

following procedures are recommended for collection and processing of serum. The birds are

exsanguinated by bleeding from the heart under

negative pressure and the whole blood of three

to five birds is pooled in a large sterile prescription bottle. The blood is allowed to clot at

room temperature for about an hour. The clot

is loosened from the walls of the container and

subsequently refrigerated overnight. The next

morning the serum is decanted and clarified by

centrifugation for 15 minutes at 2,500 r.p.m.

It is then drawn off and two to three lots pooled

together and refrigerated. As soon as possible

the individual pools are tested for RSV antibody using the different subgroup viruses.

Antibody negative pools are combined, passed

through a sterilizing filter, dispensed in small

aliquots in 100-ml. prescription bottles, and

inactivated at 56° C. for 30 minutes. They are

then tested for sterility and frozen until used.

Filtration should be done through a filter with

a pore size of 0.2,tL or less (Seitz S3 or Millipore

GS filter), since Pseudomonas or other contaminants of small size are known to occur in

blood or trypsin and may pass through filters

of larger pore diameter.

Some commercially produced agamma chicken sera have also been found to be free of RSV

inhibitory factors ; such sera can be used when

supplies of antibody-free sera are not available.

For use in Marek's disease studies, the same

or even more stringent precautions need be

taken to obtain antibody-free serum. Because

of the rapid spread of the disease at an early

age strict isolation procedures are required to

produce infectious-free stock. Agamma sera

have not been tested, but presumably they are

as useful as in the studies on LL.

Sodium bicarbonate.—Twenty-eight grams of

sodium bicarbonate (NaHCO,), anhydrous, are

dissolved in 1 1. of w^ater, and the liquid is dispensed into flasks or bottles. The solution is

then sterilized by autoclaving at 15 p.s.i. for 15

minutes. Water used in any cell-culture medium

or reagent should be as highly purified as reasonably possible. A satisfactory procedure is to

soften the water, distill it, and finally deionize

it through a mixed bed resin.^
Antibiotics.—A penicillin-streptomycin mixture is presently (1971) being used at a concentration of approximately 100 units and

micrograms, respectively, per milliliter of final

culture medium. The stock solution is prepared

as a thousandfold concentrate, 1 ml. of which

when added to 1 1. of culture medium gives the

final concentration per milliliter. One vial of

penicillin G potassium (2x10" units) and four

vials of streptomycin sulfate (5 g./vial) are

used to prepare 200 ml. of stock solution. About

5 ml. of sterile water is injected aseptically into

each vial and the vials shaken until the antibiotics dissolve. The vial contents are then removed with a needle and syringe and brought

up to a volume of 200 ml. with sterile water. The

solution is then filtered through a Seitz SI pad

and dispensed in 5 to 10 ml. amounts in screwcap vials that are stored frozen until used. (Filtration is an optional procedure utilized to help

insure sterility,)

Other antibiotics have been used periodically

for more specific control of contamination.

These include the following with their respective stock concentrations.

Mycostatin—mold and yeast control 5,000 units/ml.

Amphoterricin B—yeast control 500 units/ml.

Polymyxin—control of gram negative

bacilli 2,000 units/ml.

Terramycin—control of pleuropneumoniaHke organisms (PPLO) 5,000 nnits/ml.

Final working concentrations are a 1:100 dilution in the final culture medium.

Phosphate buffered saline base.—bOx concentrate, less NaCl

KCl 10 g.

KH2PO4 10 g.

Na^HPO^ 60 g.

a>0 1,000 ml.

All ingredients are dissolved in the water,

dispensed in multiples of 20 ml., and stored

frozen.

Trypsin,—Stock solutions of both 1.0 and 2.5

percent are used and are prepared as follows.

Either 10 g. (for 1.0 percent) or 25 g. (for

2.5 percent) of trypsin 1-300 '" are added to 1 1.

of phosphate buffered saline (reagent A, p. 5).

The mixture is stirred overnight at 4° C. on a

magnetic stirrer and then filtered through 1 to

2.5 cm. of celite filter aid." The solution is then

final filtered for sterility through an S-3 Seitz

pad (excessive foaming should be avoided), and

the filtrate is dispensed in 5 to 10 ml. aliquots

and frozen. Trypsin is sometimes variable in

its activity; therefore, if desired, assays of

trypsin activity for each new preparation can

be done. Reference is made to Erlanger and

coworkers (12) for details.

Agar.—Difco purified agar^ or Consolidated

Laboratories ionagar #2 " have been used with

success. Concentrations of 1.8, 3.0, or 3.6 percent are prepared by adding 18, 30, or 36 g.,

respectively, of agar to 1 1. of water. The mixture is boiled with constant stirring or heated

in an autoclave for 10 minutes to dissolve the

agar; the solution is then autoclaved at 15 p.s.i.

for 15 minutes. The agar is melted in a boiling

water bath just before use.

Working Media Preparation

and Solutions

The media preparations used in this laboratory are listed below with the amount and type

of stock solutions needed to prepare small volumes of 100 to 500 ml. and larger volumes of

1 to 6 liters.

Stock solution For volumes For volumes

of 100-500 ml. of IS I.

Milliliters Milliliters

Medium A.i—Base medium for chick embryo fibroblast

(CEF) and duck embryo fibroblast (DEF) growth and

maintenance,

FIO (10X)2_.__„_._,___...-„_^- 10.0 800.0

199 (10X)2,„___.„._.___.____-,- 8.0 240.0

TPB_ „--^ - 10.0 300.0

Pen-Strept__-_--.--_--- -2 6.0

Mycostatin --.__.-. 1.0 30.0

Water._,__ 162.0 4,860.0

Medium B.i—Base medium for chick kidney cell growth

and maintenance.

BME(IOX).„,--.-_--_-..----=- 40.0 500.0

TPB ___-_---. ._--- 49.6 620.0

Pen-Strept __....___» 1.0 12.5

Mycostatin^ 2.6 32.0

Water . -_ 360.0 4,500.0

Medium C.i—Hard agar base, NP test.

FIO (10X)_..„ __^_. 16.0 160.0

199 (lOX) „__ -- 12.5 125.0

TPB. 17.5 175.0

Pen-Strept_ - .3 3.0

Mycostatin -^--- l-S 15.0

Water .„ 114.0 1,140.0

Medium DJ—Hard agar base, RSV assay.

F10(10X)_„. 5.0 100.0
Stock solution For volumes of 1-6 I.

of 100-500 mL Milliliters

Milliliters For volumes

Medium D—Con.

199 (10X)„„_..._„..„.„,„.„._.„.„., 5,0 100.0

TPB„...„_.^„.„.„-„_„_...„„ _,,.„_ 14.0 280,0

Pen-Strept...„...„, „„„.,,..„_„„ .1 2.0

Mycostatin„„„^_ _..„_„._.__.. _ .6 12.0

Water„.„____„.„.^.„„,_.._ 40.0 800.0

Medium E.—Growth medium for CEF and DEF.

Medium A-,.. -.-,.„,.... „ ^..... _ 100 2,000

Calf serum..^^^,.._.„- ,. ,„....... 4 80

2.8 percent bicarbonate._.„.^._„ 4 80

Medium F. -Hard agar overlay, NP test.

Medium C. .„. _.. _.,. ^, _.. 60 600

8.6 percent agar „ „ _.... „ ,„„.„„ 40 400

Bovine amniotic fluid „._^....„ 20 200

Calf serum_._„, ._..„„._„. 2.4 24

2.8 percent bicarbonate„.,„ 5,0 50

Medium G.—Hard agar overlay, RSV assay.

Medium D^.^...^..........,. „-.- 60 600

1.8 percent agar __„„__,.._„„. 40 400

Calf serum .„„_._^_,..__. 5 50

2.8 percent bicarbonate „_.... 4 40

Medium H.—Feed medium for agar overlay plates.

199 (10X)___„_._„„,_„„„„„,.. 10.0 100.0

2.8 percent bicarbonate. _„-...,-,» 2.0 20.0

Pen-Strept„__ ..,„.__.,._ ^.... -. ,1 1.0

Mycostatin.. _^ „„.„_„„_„„„„..„«„ .5 5.0

Water.„.„_„„„^ _„.. 90.0 900.0

Reagent A.^~-Phosphate buffered saline (PBS)

PBS base (50X).„_,,„„^_„„..._-„. 20.0 60.0

NaCL_„-„-„_„-.„„_„_._-._...„ 4 8.0 424.0

Water„.„„.„ ..„__„„_„„„ „„^_„„„, 980.0 2,940.0

Reagent B,—Trypsin (0.05 percent) for monolayer dispersion.

Trypsin (2.5 percent)„ __„_ ..„„-„ 1.0 10.0

Reagent A (PBS)„.,......-.--^-^^ 49.0 490.0

Reagent C,-~Trypsin (0.125 percent)^ for embryo cell

dispersion.

Trypsin (1.0 percent), ._„„„„ 10.0 100.0

Reagent A (PBS).„,_._. _._-._.-. 70.0 700.0

1 Various concentrations of NaHCOs, bovine serum, and

other additives are added in the different tests. These are

indicated later,

2 CEF have been found to grow better in a mixture of

medium 199 and medium FIO; also focus formation by RSV

was better and the titer higher in this medium (unpubhshed

data of J. J. Solomon and K. Giatt). It has therefore been

adopted for most fibroblastic cultures in our Laboratory.

s The BOX concentration is thawed and added with

sodium chloride to the water. The ingredients are thoroughly

mixed and dissolved, and the solution is dispensed in bottles.

The solution is autoclaved for 15 minutes at p.s.i. The pH

should be between 7.0 and 7.4*

4 Grams instead of milliliters.

^ Trypsin at 0,25 percent is also satisfactory for embryo

cell dispersion. It is prepared by diluting either the 1.0 or

2,5 percent trypsin.

Cell Preparations

Preparation of Chick Embryo

Primary Cells

Chick embryo primary cells are prepared in

the following manner. The outside of the shell

of a 9- to 11-day-old embryonated egg is disinfected by immersion in 70 percent ethyl alcohol for 5 minutes. The shell above the air cell

is then cut away with scissors, and the embryo

is lifted out of the eggy decapitated, and placed

into a 125- or 250-ml. erlenmeyer trypsinization

flask containing about 30 ml. of sterile PBS

(reagent A). Usually 3 to 5 embryos from the

same dam are pooled in one flask. The embryos

are washed at room temperature on a magnetic

stirrer for approximately 3 minutes. This is

repeated with fresh PBS until the saline remains fairly clear. The embryos are then macerated by holding the flask in a tilted position and

allowing the stirring bar to rotate at a moderately fast speed so that it forces the embryo

against the flask. This operation requires about

1 to 3 minutes per flask. It avoids opening the

flask for mincing the embryos with scissors or

spatula and thug decreases the chance for contamination. In general, the smaller the tissue

pieces, the greater the final cell yield. The

macerated tissue is washed for 2 or 3 minutes,

the tissue fragments are allowed to settle, and

the supernatant saline is removed. The procedure is repeated until the saline remains fairly

clear. After the final wash, about 100 ml. of

warm trypsin (37° to 48° C.) (reagent C) are

added to each flask, and the flask contents are

stirred slowly on a magnetic stirrer for 30 to

60 minutes. For optimum trypsinzation the temperature should be at 37° to 48° C. during this

period. The heavier particles are allowed to

settle for 1 to 3 minutes and supernate is removed and centrifuged at 500 r.p.m. for 15

minutes or 1,200 r.p.m. for 3 minutes. The cell

pellet is resuspended in medium E and mixed

by aspirating or with a vortex mixer, and cells

are counted^—care should be taken not to include the nucleated red blood cells. If the cell

suspension is heavy (greater than 3 to 5 X 10''

cells/ml), it should be diluted (1:5 to 1:10)

' For use of hemaeytometer, see Appendix, p. 18.
and recounted- The cells are then plated in

medium E at concentrations of 1 X 10* cells in

8 to 10 ml. for lOO-mm.-diameter Falcon plastic

tissue-culture dishes or 2.0 X 10" cells in 20

to 25 ml. for 150-mm.-diameter dishes. The

latter size is more efficient in cost and labor.

Cells of known RSV sensitivity phenotype

must be used in studies on lymphoid leukosis.

Sensitivity is determined by the following procedure. One million primary cells are seeded in 4

ml. of medium E in 60-mm.-diameter petri

dishes, and the plates are incubated overnight at

37° C. The medium is removed and replaced

with 4 ml. of fresh medium to eliminate cells

that did not adhere and grow. The cells are then

challenged with 0.1 ml. of RSV from each subgroup, diluted in medium A to contain 10^ and

10^ focus-forming units (FFU) of virus. Two

plates are challenged with the low dose and two

with the high dose from each subgroup, and 2 to

18 hours later the liquid medium is removed and

5 ml. of agar overlay (medium G) added. The

plates are reincubated for 3 days and 2 to 3 ml.

of feed medium H added over the agar. The cells

are observed 4 to 7 days after infection for the

presence of mature foci and a count of foci is

made during this period. (For details on development and counting of RSV foci, see p. 7.)

With the high dose of virus, sensitive cells

will show signs of RSV infection within 3 or 4

days. These appear as high acidity compared

with noninfected controls and the presence of

RSV foci. Some evidence of embryo sensitivity

can thus be rapidly obtained from the high-dose

challenges and sensitivity primary cells selected

for experimental purposes shortly threafter.

The plates challenged with 1,000 FFU should

be held 4 to 7 days before the foci are counted.

This gives Quantitation of the cell sensitivity

to RSV. Cells showing a RSV focus count ^0.1

of the infecting dose are considered to be resistant to RSV, owing to the presence of LL

viruses or inherent genetic factors. Genetic resistance can be distinguished from viral resistance by lack of transmission with cell-free

fluids from the monolayer.

Preparation of Duck Embryo

Primary Cells

Duck embryo fibroblasts are used in the study

of Marek's disease and in the NP test for LL

viruses. They are prepared in the same manner

as the chick embryo primary cells with the exception that 13- or 14-day-old embryos are used.

Cells from all sources tested have been sensitive

to the herpes virus of MD; thus primary-cell

sensitivity tests are not necessary.

Preparation of Chick Kidney Cells

Chick kidney cells are also used in the study

of MD. They can be obtained from embryos,

young chickens (1-3 weeks old), or adult birds.

The general procedure is to remove the kidneys

aseptically, wash them with PBS in a small

beaker, and mince them with scissors. An alternate procedure is to inject heparin intravenously just before the chicken is killed and immediately after to perfuse the kidney with 0.05

percent trypsin via the common iliac vein. The

minced kidney is then trypsinized for two cycles

of 5 to 10 minutes v/ith 0.05 percent trypsin,

and the dispered cells are discarded. Several

additional trypsinization cycles are carried out,

and the cells are collected and plated in medium

B containing 0.084 to 0.112 percent bicarbonate

(3 to 4 ml 2.8 percent/100 ml.) and 5 percent

fetal bovine serum. The serum content is reduced to 2 percent after a confluent layer is

obtained, 1 to 3 days after plating.

For tissue-culture procedures in general, the

textbook by Parker {25) and the laboratory

manual by Merchant and coworkers {22) are

recommended. In our Laboratory we have

found that changing growth medium on the

monolayers on alternate days gives better cell

growth and maintenance. It is also very important to maintain the pH near neutrality and

this often requires a neutralization of the acid

produced by the cells on the day after the

medium change. This is done by the addition

of enough (0.1 to 0.5 ml.) sterile 2.8 percent
NaHCOa to the plates to bring the color back

to that of fresh medium. It is essential that cells

be observed frequently and that overcrowding

be avoided by cell transfer or the use of maintenance medium after confluent monolayers are

achieved. For maintenance purposes, the serum

content in the grov^th medium can be reduced

to 1 to 2 percent or 10 percent bovine amniotic

fluid can be added to the regular media formulations.

VIRUS PREPARATIONS

Rous Sarcoma Virus (RSV)

Tumor Induction and Harvest

Stocks of standard RSV of high potency may

be propagated in cell culture in vitro or extracted from tumors induced in chickens in vivo.

Stocks of virus with titers of approximately

lO'^' to 10" FFU per milliliter are readily obtained by either method. For the in vitro propagation of RSV, Bader's procedures (2) are

recommended. The following is a brief description of the induction of tumors in chickens and

the subsequent extraction of virus by Moloney's

method (28).

Susceptible chickens B to 5 weeks of age, free

of LLV, are inoculated intramuscularly in the

pectoral muscle or subcutaneously in the wing

web with 0.2 ml. of RSV containing 10 ^ or 10^

infectious units of virus. Beginning at 5 days

after inoculation, the chicks are examined daily

for tumor growth at the site of inoculation. The

growth of the tumors is very rapid ; the tumor

tissue should be harvestable between the

seventh and 14th day. During this time the

pectoral region of chicks inoculated at this site

will enlarge to several times its normal size,

and tumors induced in the wing web should

approach 15 to 20 mm. in diameter or larger.

The tumor growth should not be allowed to

progress to the hemorrhagic, necrotic, or ulcerative state, which will occur if held too long.

The tumor masses should be harvested using

aseptic techniques ; care should be taken not to

include hemorrhagic and or necrotic areas. The

excised tumor material should be kept cold, preferably in containers on ice. It is pooled, minced,

frozen, and stored at —70'' C. as soon as possible after harvest.

Partial Purification (Moloney's method

(23)) : The frozen tumor mince is thawed at

room temperature and made up to 6.6 percent

(wt,/v.) with 0.15M potassium citrate containing 1 mg. percent hyaluronidase. The suspension is allowed to digest at room temperature

in a Waring Blender with intermittent mixing

(15 seconds at 10-minute intervals) for 1 hour.

At the end of this period, it is homogenized

thoroughly for 3 minutes. The homogenate is

centrifuged in the cold at 2,300 X g. for 20

minutes, and the supernatant fluid is carefully

pipetted off; the top fatty and lower sediment

layers should be avoided. This is designated the

Si fraction. The centrifugation (2,300 X g. ior

20 minutes) is repeated, and the resultant fluid

is designated S2. The S2 fraction is centrifuged

at 18,000 X g. for 1 minute; the supernatant

(S3) is poured off and retained. The S3 fraction

is passed through a bacteriological niter, vialed,

sealed, and stored at —70° C.

Assay of RSV

The first step is to obtain RSV sensitive primary cells. This can be determined from the

results of the primary challenge or from a past

history of RSV sensitivity of embryos from a

particular dam. However, the sensitivity of the

cells must also be confirmed during the particular experiment in progress. The CEF monolayers are prepared by decanting the fluid layer

from the RSV sensitive primary cells into a

container fitted with a gauze cover to reduce

splash. Six to ten ml. of warm (37^ C.) 0.05

percent trypsin are added to each plate, and

the plates are reincubated at 37° C. for 10

minutes. This procedure usually loosens most
8

of the cells. The remaining attached cells can

be removed by forcing* the trypsin-cell mixture

against the bottom of the petri dish through a

10 ml. pipette or by scraping with a sterile

rubber policeman. When all cells have been removed, the suspension is transferred to a large

centrifuge tube. The cells from several plates

are pooled together in the tube and centrifuged

at 1,200 r.p.m. for 3 minutes. They are then

resuspended in medium E (about 7 to 10 ml.

for each plate of cells added to the pool) and

counted in a hemacytometer. One million cells

are then added to a small petri dish, and the

volume is brought up to 4 or 5 ml. with medium

E. The plates are gently shaken until the cells

are evenly distributed and then placed in the

incubator.

The virus is now diluted with feed or growth

medium-—serial tenfold dilutions should be

used. All manipulations of the virus before addition to the GEF cultures should be done in the

cold. One-tenth ml. of each dilution of virus is

then added to each of two petri dishes prepared

above, and the plate contents gently but thoroughly mixed. Next morning the fluid medium

is removed and replaced with 5 ml. of agar

overlay medium G. The interval between infecting the cells and overlaying the agar should

not exceed 18 hours, since virus may then be

produced by the cells and the titer will appear

to be higher. Four days after infection, 2 or 3

ml. of feed medium H are added to each plate.

Gells infected with BSV show no visible

change during the first 2 days. On the third day

they become granular and also appear darkened. Glusters of rounded up cells can be seen

throughout the plate by the fourth day, and

mature foci of rounded up cells develop shortly

thereafter (fifth day). The sixth and seventh

day cells show the foci clearly. Development of

foci is asynchronous; therefore, foci usually

should be counted as late as 7 days after infection. With some virus strains and under certain

culture conditions, the whole cycle of focus development is shortened and counting can be

done by the fourth or fifth day. Some cells are

more sensitive to the virus than others ; hence,

careful observation of the plates is necessary

since sensitive cells are rapidly destroyed by

high concentrations of virus.

Counting of the foci is done with an inverted

microscope. The center piece on the stage is

removed and a counting grid^^ installed in its

place. The foci on a representative fraction of

the area of the plate are counted, and the total

foci per plate are caluclated by multiplying this

number by the reciprocal of the area counted.

The procedure is valid only when uniform distribution of the foci are obtained. Where only

a few foci are present, the foci on the entire

plate are counted. Since each focus is presumably initiated by a single infectious unit, a

count of foci gives a titer of the RSV preparation. However, Rubin (SI) has suggested the

addition of 1 log to the titer, since he found that

of 10 infected cells only one actually goes on to

initiate a focus,

Lymphoid Leukosis Virus (LLV)

Propagation,—Strain RPL-12, subgroup A

virus has frequently been used in the study of

lymphoid leukosis and related neoplasms in vivo

and as an RSV-resistance-inducing virus in

tissue culture. Virus propagation at this Laboratory has primarily been done in vivo (5).

For tissue-culture procedures of propagation

of LL virus, the papers by Rubin (33) and

Bader (2) are recommended.

End point assay.—The end point assay of LL

virus is done by the RIF procedure. This is

detailed in the section on virus assay, lymphoid

leukosis, under RIF test.

Marek*s Disease (MD)

Propagation of the agent has been done primarily in vivo because of the highly cell-associated nature of the agent and the absence until

recently (9, 38) of in vitro techniques. Details

on the in vivo propagation of two strains can be

obtained by reference to Witter and coworkers

(45).

In vitro propagation can be done on either

chick kidney or duck embryo cells. The latter

procedure will be described in detail. DEF primary cells are trypsinized in the same manner

as GEF primary cells in the RSV assay. The

cells are then counted and plated at 3 X 10^

cells per 100-mm. petri dish in medium E and
incubated overnight. One or more plates each

are- then inoculated with 0.2 ml. of tissue infected with the agent to be propagated. Whole

blood, buffy coat, tumor, or other tissue can be

used. The plates are reincubated for 1 to 24

hours, the inoculum gently washed off with

PBS, and the plates maintained under liquid

for up to 12 days. Herpes virus plaques began

to appear between the fifth and eighth day after

infection, and the cells can be harvested shortly

thereafter. The cells are removed by trypsinization, resuspended in medium A containing 15

percent calf serum and 5 percent DMSO, and

vialed in 1-ml. quantities at a concentration of

5 to 10 X lOVml. Cells in DMSO must be kept

on ice. The vials are then sealed, the temperature immediately lowered at a rate of about

1"" C./minute down to —40°, and the vials stored

in liquid nitrogen.

Assay of the agent is done by titering the

infected cells on DEF monolayers in the same

manner as in the propagation of the agent.

Plaque morphology is described under Marek's

disease in the section on virus assay. Increases

in titer can be obtained by repeating the propagation procedure with the infected DEF as

the inoculum.

VIRUS ASSAY

Lymphoid Leukosis

There are a number of in vitro assay procedures for LL virus, but only the more commonly used or newer techniques will be described in detail. These include the RIF,

COFAL, and NP activation procedures. The

latter is the result of work by many investigators but in particular the work of Hanafusa

and others {15, 16, iO) on the defectiveness of

RSV and the helper function of leukosis viruses.

Rispen and coworkers (30) established an in

vitro system for this assay procedure by cocultivating NP CEF with duck embryo cells. The

addition of test material containing LL virus

to this system activates the NP cells to produce

RSV. The presence of RSV in the cell-free

culture fluids can then be detected by one of

several techniques to confirm the presence of

LL virus in the test sample. The NP activation

test is rapid and simple after the preparation of

NP cells and compares in sensitivity to the RIF

and COFAL assays (SO). Information on other

procedures for the assay of LL virus can be

found in the references cited or in other literature. For information on the use of fluorescent

staining techniques in studies on LL virus (7,

19, 20, 21, 26, ÍS) are suggested.

Resistance Inducing Factor

(RIF) Test

The RIF test can be initiated on either primary or secondary RSV sensitive CEF. Cells are

plated at a concentration of 5 to 10 X 10^

primary or 2 to 3 X 10^^ secondary cells in

100-mm. petri dishes, and 0.1 ml. of test material (serum, plasma, tumor extract, or similar

material) is added to the plates shortly thereafter (up to 18 hours after plating). Duplicate

plates should be inoculated with each sample

and positive and negative controls included in

each test. Negative controls are of two types,

i.e., plates that are uninoculated and plates inoculated with known LL virus-free chicken

serum. Positive controls are inoculated with

serial dilutions of standard LL virus of all

known subgroups or with dilutions known to

encompass the endpoint titer of the standard

viruses.

The plates are incubated for 3 days, the fluid

medium is discarded, and the cells are removed

by trypsinization. After centrifugation the cells

are resuspended in growth medium E and

counted. One million cells are then added to

each of four small assay plates (60-mm. diameter), and 2 to 3 X 10' cells added to one or

more 100-mm. plates. The larger plates are

immediately reincubated for 3 or 4 days for

use in the second transfer (see below). Pseudotype RSV from each subgroup is diluted to

contain about 10^ FFU/ml. and 0.1 ml. is used

to challenge the small plates, one subgroup per

plate. On the following day, the fluid medium

is removed and replaced with agar overly medium G. Three days later, 3 ml. of feed medium H
10

are added to each plate, and foci are counted

4 to 7 days after infection. This constitutes the

first transfer and first RSV challenge.

Three or four days later the second transfer

and challenge are made with the large plate(s)

of cells from the first transfer in the same manner as with the originally infected large plate,

i.e., trypsinize, count, and seed the cells into

four small plates for subsequent RSV challenge

and into one or more large plates for the next

transfer. This procedure is continued until no

further increase in resistance is found with the

higher dilutions of control LL virus. Two to

four transfers are usually adequate to detect

most virus in test materials, but several additional transfers are desirable and should be

conducted if the condition of the cells permits.

Challenge with RSV may be restricted to alternate transfers. A reduction ^ 1 log (^ 90 percent) in the number of foci on test plates compared with the negative controls is positive for

the presence of LL virus.

The titer of an unknown can be estimated by

reference to a standard LL virus titration curve

(endpoint assay of LL virus), which is done in

the following manner : RSV sensitive CEF are

prepared as in the RIF test and inoculated with

serial tenfold dilutions of standard LL virus.

The cells are passaged at 3-day intervals, as in

the RIF test, and challenged at each passage

with RSV from the same subgroup as the LL

standard virus. The procedure is continued until

no further increase in resistance is found with

the higher dilutions of virus. The endpoint is

usually obtained after 2 to 4 transfers, but this

must be determined for each strain of virus.

The data are analyzed by plotting relative sensitivity (RSV focus count of sample/RSV focus

count of negative control) against log virus

dilution for each passage (fig. 1). The titer of

the standard virus can then be determined from

the graph. In the figure shown a titer of 10^

infectious units/0.1 ml. (lOVml.) is obtained,

and in this instance the endpoint is reached

after only two passages.

From a standard LL virus curve, such as

that shown in figure 1, the titer of an unknown

can then be estimated. For example, the titer of

a sample tested undiluted and inducing ^ 1 log

resistance on the first challenge would be between 10 6 and 10 ® infectious units per milliliter.

Failure to obtain resistance on the first challenge would indicate a titer at least 4 logs less

than the standard LL virus if resistance was

obtained on the second and third challenge. The

comparative interference assay of Vogt and

Rubin {JfJf) can also be used to determine the

titer of an unknown LL virus. In this procedure

the titer of a standard virus is first determined

by the endpoint assay (as above). A sample of

the standard and the unknown are then diluted

serially and used to inoculate different chick

embryo cultures. The cells are transferred and

challenged 3 days later, and the level of resistance of each dilution is determined. The relative sensitivity to RSV is then plotted as a function of the sample dilution on a logarithmic

scale. Parallel straight lines are obtained, the

curve of the unknown being displaced horizontally from that of the standard. This displacement corresponds to a dilution factor and the

titer of the unknown is less than or greater

than the titer of the known by this factor.

Complement Fixation for Avian Leukosis

(COFAL) Test

The COFAL test is performed in two phases:

(1) LL virus propagation or antigen production in CEF cells, and (2) assay of specific

complement fixing antigen. In addition to the

standard complement fixation (CF) test reagents obtainable from numerous commercial

sources,'^' the COFAL test requires the following: (1) LL virus susceptible chicken embryo

fibroblasts; (2) stock COFAL positive antigen,

which may be obtained as extracts from RSV

induced chicken tumors, RSV or LLV infected

CEF, or RSV induced hamster tumors ; and (3)

stock positive antiserum (hamster), which may

be produced by the inoculation of newborn or

weanling hamsters with RSV or RSV induced

hamster tumor cells and the subsequent collection of serum from tumorous hamsters. However, since this procedure is time consuming

and the titer and specificity of the serum from

each hamster are highly variable, it is strongly

suggested that antiserum be purchased from a

commercial source.'^

The test samples are inoculated onto primary

or secondary CEF and held in culture for ap-
11

1.0

0.8

c

wo

>

0.6

0.4

0.2

o Ist CHALLENGE

A 2nd CHALLENGE

D 3rd CHALLENGE

Virus Dilution (-Log in )

FIGURE 1.—RIF assay curve of RPL12—LSI, Lymphoid leukosis virus.

proximately 21 days with cell transfers at 3- to

5-day intervals, as in the RIF test procedure.

Positive controls, consisting of CEF inoculated

with known LL viruses from the various subgroups, as well as uninoculated control CEF

cells are required for each test. Although some

of the rapidly propagating viruses each endpoint titers in 12 to 14 days, other slower growing types require somewhat longer. At the end

of the culture period, all but 0.2 to 0.3 ml. of the

fluid is removed and the cells are scraped free

of the culture surface with a rubber policeman

and suspended in the retained fluid. The cell

suspension is frozen and thawed three times

(alcohol-dry ice) or sonicated to rupture the

cells and then clarified by low-speed centrifugations; the clear fluid is used as antigen in the

complement fixation test.
12

The microtiter^ system (S6) is highly recommended for use in the complement fixation

phase (4) of the COFAL test. It has been shown

to be accurate, and highly reproducible results

can be obtained {S6), In addition, a small volume of reagent aliquots (0.25 ml.) is required

and three to six sample dilutions can be made

in one operation; hence, other standard CF

methods appear obsolete.

The CF phase of the test is performed as

follows: The test sample is diluted twofold

across a series of wells. Anticomplementary

(AC) controls are included as the two highest

concentrations of test material. The 0.025-ml.

loops and dropper pipettes are used for this

dilution. Veronal buffered saline, pH 7.3, is

used for all reagent diluent. Antiserum (hamster, 4 units in 0.025 ml.) is added to all test

samples except the AC controls, in which

veronal buifered saline is used. Complement^

(2 exact units in 0.025 ml.) is added to all wells,

test samples as well as AC controls. The plates

are gently shaken, (precautions must be taken

not to splash the dilution over from well to

well) and held static at 4° C. overnight (approximately 16-18 hours). The following morning, sheep erythrocytes ^ ^ collected or stored in

Alsever's solution (i) are washed three times

in veronal buffer, resuspended to a 2 percent

concentration, and sensitized for 15 minutes at

room temperature with an equal quantity of

diluted hemolysinJ to contain 2 units per 0.025-

ml. aliquot; 0.05 ml. of these sensitized sheep

erythrocytes are added to each well. The plates

are carefully sealed with wide transparent cellophane tape, shaken vigorously, and held at

37° for 1 hour with a shaking after the first

10 minutes of incubation. The plates are then

held in a static position at room temperature

for 1 hour and read for fixation. Fixation, or a

positive reaction for LL antigen, is indicated by

the absence of lysis of sheep erythrocytes. The

following controls are always included with each

test: (1) Standard positive antigen AC control,

(2) standard positive antiserum AC control,

(3) complement activity controls of three concentrations—2,1, and 1/2 units, (4) erythrocyte

fragility control, and (5) known positive system control.

Nonproducing (NP) Cell Activation

Test

The procedure and reagents found most suitable for the NP test are outlined below. Two

phases are required for this procedure : (1) production of NP cells and (2) assay of LL virus.

(1) NP cell production

a. Materials

(1) CEF as in RIF and COFAL

procedures.

(2) DEF

(3) BH-RSV (Rav-1) or other

BH-RSV pseudotypes.

b. Procedure

Primary or secondary cells are

plated at a concentration of 1 to 2 X

10^ CEF and 10 ^ DEF in 60-mm. petri

dishes. The cells are infected while in

suspension with 0.2 ml. of serial twofold dilutions of RSV, duplicate plates

for each dilution. Plates with 50 foci

or less are desired for cloning, and

appropriate dilutions of RSV to be

used can be determined by titering the

virus beforehand. After overnight incubation, the cells are overlaid with

5 ml. of agar overlay medium F containing high titer chicken antiserum

prepared against the RSV being

cloned. Five days later a second overlay of 2.5 ml. is added onto the previous agar layer. Ten to eleven days

after inoculation, about 25 isolated,

large dense foci without degenerate

centers are selected and the agar immediately above is cut with a sterile

cork borer and removed aseptically.

Contact between foci must be avoided

to prevent possible cross contamination with RSV. The cells are then trypsinized by adding a small amount of
13

1 percent trypsin (diluted 1:20 to 1:50

with PBS) and harvested with a pasteur pipette. The trypsin action is reduced by cooling or by the addition of

about 0.5-ml. calf serum per focus harvested. The cells obtained from each

focus are plated on freshly prepared

secondary DEF, one focus per 60-mm.

plate containing 1.0 to 1.2 X 10' DEF

in medium E. The cells are then subcultured at 7- to 10-day intervals, and

at each subculture supernatant fluid is

tested for the presence of cell-free

RSV by either in vitro cell assay or

in vivo chorioallantoic membrane

(CAM) assay. Only those clones free

of infectious RSV are retained and

used in the production of NP cells.

The in vitro assay for cell-free RSV

is done by adding 2 to 4 ml. of culture

fluid to RSV sensitive, RIF-free CEF,

followed by an agar overlay 18 to 24

hours later. Three days after infection,

3 ml. of feed medium is added, and

3 or 4 days later the monolayer is observed for foci.

CAM assay is done by inoculating

0.2 ml. of culture fluid onto the CAMs

of 9- or 10-day-old RIF-free, RSVsensitive embryonated eggs. The

CAMs are removed and examined for

pocks 8 or 9 days later.

Cells that are found to be nonproducing are then propagated through

several passages until the number of

cells is adequate for large-quantity

storage. At the start of each passage,

fresh secondary DEF are mixed with

the NP cells to obtain confluent foci

at the end of the passage and increase

the number of cells. The ratio is generally determined by visual inspection

of the cell monolayer at intervals before passage. A 1:1 ratio of NP to

fresh DEF cells has been found to be

satisfactory during many of the passages. About five passages are required

to increase the number of cells sufficiently to obtain storage quantities.

Medium E with 1 percent calf serum

or 2 percent bovine fetal serum plus

10 percent bovine amniotic fluids

(BAF) has been found to be an excellent maintenance medium for cells

24 hours after transfer.

At the end of the propagation period, the NP cells are harvested by

trypsinization and resuspended at a

concentration of 5 to 10 X 10^ per ml.

in medium E containing 20 percent

serum, 10 percent BAF, and 5 percent

DMSO. (Cells in DMSO should be kept

in ice.) They are then vialed in 1-ml.

quantities and frozen by lowering the

temperature from 5° to —40° C. at

the rate of 1° per minute. The sealed

vials are then stored in liquid nitrogen.

(2) Assay for LL virus (NP cell activation

and RSV assay)

Direct Activation Test

a. The NP cells are removed from

liquid nitrogen storage and rapidly

thawed in water at 37° C.

b. Fresh secondary DEF, NP cells, and

fresh secondary CEF (type C/0, LL

virus free) are plated at 1.0, 3.0, and

4.0 X IOS respectively, in a 60-mm.-

diameter plate.

c. These cells are inoculated 24 hours

later with 0.2 to 1.0 ml. of test material per plate.

d. Plates are incubated overnight at

37° C, the culture fluid is removed,

and the cell monolayer is washed with

PBS. The growth medium is replaced

with maintenance medium (medium

E, 1 percent calf or bovine fetal serum,

and 10 percent BAF).

e. The fluid is harvested on the fifth,

seventh, or ninth day, and the RSV is

assayed by either CEF or CAM assay.

f. The following controls are required

during this test procedure:
14

Gomponent

(1) Monolayer of NP cells alone.^

(2) Monolayer of NP and DEF

cells. ^

(3) Monolayer of NP and CEF

cells,^

(4) Monolayers of NP cells infected with dilutions of LL virus

of the known subgroups (span

endpoint dilutions) .^

Purpose

To assure the nonproducer status of the

NP cells.

To test the DEF for congenital or adventitious infection with LL virus.

To test the CEF for congenital or adventitious infection with LL virus.

For positive control on the sensitivity of

the NP system.

^Culture fluid from controls (1), (2), and (3) must not contain RSV.

- Culture fluid from control (4) snoula contain RSV through the endpoint LL virus

dilutions.

The NP cell activation test can also

be used to test for the presence of LL

virus in cultured CEF. This procedure

is frequently used in the EIF test to

determine the infection status of the

embryo. This is done by adding the

CEF being tested to fresh secondary

DEF and NP cells and assaying the

culture fluid for cell-free RSV (as in

steps d and e of the direct activation

test.)

Marek's Disease

Marek^s disease virus can be assayed in cell

culture by the use of either duck embryo fibroblasts or chick kidney cells. The chick kidney

system appears to be somewhat more sensitive

than the duck cell system. When virus assays

are to be conducted on chickens to be sacrificed,

direct culture of the kidney is preferred because

it is considered somewhat more sensitive.

For chickens that cannot be sacrificed, the

following techniques are used.

Indirect kidney cell test.—Chick kidney cells

are prepared and plated at a concentration of

5 to 8 X 10^ per 60-mm.-diameter plate in

medium B with bicarbonate and serum, as indicated under preparation of kidney cells. After

24 to 48 hours the plates are inoculated with the

material being tested (whole blood, tumor cells,

tumor extracts) and reincubated. In 3 to 24

hours after inoculation, the inoculum is washed

off with PBS and fresh medium is added. When

the cells are confluent (usually 48-72 hours) the

medium is changed to a maintenance medium

by reducing the concentration of bovine fetal

serum to 1 to 2 percent. The cytopathogenic

effect (CPE) can be observed 3 to 5 days after

inoculation and enumerated 9 to 10 days after

inoculation. The CPE is easily distinguished and

consists of rounded or elongated focal clusters

of cells against a flat monolayer of normal cells.

Positive and uninoculated controls are carried

in each experiment.

Duck embryo cell assay,—The duck embryo

cell assay system is similar to the kidney cell

system ; secondary DEF are inoculated with the

material under test, and cells are observed 7 to

14 days after infection for CPE. Secondary cells

are plated in medium E at 1.0 X 10"^' per 60-

mm.-diameter plate and inoculated up to 48

hours later, generally with 0.1 ml. of inoculum.

Higher concentrations of some inocula may be

toxic to the cells. In 8 to 24 hours after inoculation, the inoculum is washed off with PBS and

fresh growth medium is added. CPE generally

first appear in 5 to 7 days and may be enumerated 10 to 14 days after infection. During this

period the plates must be observed for cell

crowding and decreased pH. In some instances

a maintenance-type medium, medium E with 1

to 3 percent calf serum, is substituted. It is important that the cell physiology be as optimal as
15

possible. The CPE that appears is similar to

that obtained with the chick kidney cell monolayers except that there are fewer round cells

and more elongated cells. Positive and negative

controls should be carried in each experiment.

Bovine fetal serum has been found to be detrimental to the formation of CPE in the DEF

system ; hence, it should not be used.

ANTIBODY ASSAY

Lymphoid Leukosis

Antibody to LLV is measured by its reaction

with RSV and the neutralization of RSV is

determined by any of the RSV assay procedures, i.e., chick or embronated egg inoculation

and cell culture. Only the latter in vitro technique will be given in detail.

RSV of the subgroup(s) of interest are selected for the assay and diluted to contain about

10* FFU/ml. The test serum or plasma is inactivated at 56° C. for 30 minutes and 0.1 ml.

incubated with 0.9 ml. of virus at ST" for 40

minutes. One-tenth ml. is then plated on sensitive CEF and overlaid with agar on the following day. The number of foci are counted 4 to 7

days after infection. A reduction in the number

of foci of ^ 90 percent is considered positive

for Rous virus antibody. Negative sera fail to

inhibit the virus and the number of foci is

similar to that found on the control cells inoculated with virus alone. The antibody titer of

the serum is expressed as the dilution of serum

that reduces the titer of RSV by 1 log or more.

Marek's Disease

Two techniques, only recently available (§,

27, 28) have been used to detect antibody to

MD—the agar gel precipitin test and the fluorescent staining technique.

Agar gel 'precipitin test.—The agar gel precipitin test of Chubb and Churchill (<§), with

either MD infected chick kidney cells or duck

embryo ñbroblasts as the antigen, is a rapid and

simple technique for detecting antibody in sera

or plasma. Antigen is prepared by infecting cells

with MD agent and culturing the cells through

several passages until a number of plates with

confluent or heavy CPE are available. At this

point the cells are trypsinized, resuspended in

PBS at a concentration of 4 X lOV^nl. and

rapidly frozen and thawed three times. The

antigen is then ready for testing against a

known antiserum. This is done on an agar gel

slide or plate by means of the Ouchterlony technique. Agar is melted, a preservative (either

0.5 to 1.0 percent phenol or 1:10,000 merthiolate) is added when the agar is about 45'' to 48''

C. and slides or plates are prepared. Small circular wells are then cut in the agar in a symmetrical pattern around a central well with a

cork borer or patterned cutter and serum and

antigen added to adjacent wells. The agar slide

is observed at 24 and 72 hours for the formation of precipitin bands between serum and

antigen, which indicates a positive reaction.

Fluorescent staining technique.—First the

antigen is prepared by infecting 24- to 48-hour

DEF or CK cells on coverslips with MD agent.

The cells are cultured for 3 to 5 days after

infection, fixed in acetone at 4° C, and air

dried. The antigen thus prepared is ready for

use or the cells can be stored frozen for future

tests. The test for antibody is made by flooding

serum or plasma diluted 1:20 in PBS onto the

cells and keeping it at room temperature for 30

minutes. The cells are then washed in PBS for

15 minutes, flooded with anti-chicken conjugated globulin,^ and again incubated at room

temperature for 30 minutes. The wash procedure with PBS is repeated, and the cells are

rinsed with distilled water and mounted on a

slide in glycerin buffer or some other nonfluorescing mounting medium. The slide is then

observed for staining by fluorescent microscopy.

The test correlates well with the agar gel precipitin test and appears to be more sensitive.

For additional details on the procedure the

references by Purchase {27, 28) are recommended.
16

LITERATURE CITED

(1) AHLSTROM, C. G., and FORSBY, H.

1962. SARCOMAS IN HAMSTERS AFTER INFECTION

WITH ROUS CHICKEN TUMOR MATERIAL. J.

Expt. Med. 115: 839-852.

(2) BADER, J. P.

1962. PRODUCTION OP INTERFERON BY CHICK EMBRYO CELLS EXPOSED TO ROUS SARCOMA CELLS.

Virology 16: 436-443.

(3) BERGS, V. V., and GROUPE, V.

1962. EFFECT OF ANTI-VIRAL ANTIBODY ON VIRUSCELL INTERACTIONS IN TURKEY ROUS SARCOMA

CELLS. NatL Cancer Inst. J. 29: 723-737.

(4) BRUMFIELD, H. P., BENSON, H,, and POMEROY, B. S.

1961. PROCEDURE FOR MODIFIED COMPLEMENT FIXATION TEST WITH TURKEY, DUCK AND CHICKEN SERUM ANTIBODY. Avian Dis. 5: 270-

282.

(5) BURMESTER, B. R., GROSS, M. A., WALTER, W. G.,

and FONTES, A. K.

1959. PATHOGENICITY OF A VIRAL STRAIN (RPL-12)

CAUSING AVIAN VISCERAL LYMPHOMATOSIS

AND RELATED NEOPLASMS. II. HOST-VIRUSINTERRELATIONS AFFECTING RESPONSE. NatL

Cancer Inst. J. 22: 103-127.

(6) _WlTTER, R. L.

1966. AN OUTLINE OF THE DISEASES OF THE AVIAN

LEUKOSis COMPLEX. U. S. Dept. Agr. Prod.

Res. Rpt. 94, 8 pp.

(7) CHERRY, W. B., GOLDMAN, M., CARSKI, T. R., and

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1969. CHARACTERISTICS OF TWO NEW AVIAN TUMOR

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1965. ANALYSIS OF THE DEFECTIVENESS OF ROUS

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17

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18

APPENDIX

Sources of Equipment and Media

a. Bélico Glass, Inc., Vineland, N. J. 08360;

Owens, Illinois (Kimble Products), Toledo,

Ohio 43601; and Corning Glass (Pyrex

brand), Corning, N. Y. 14832, are three of

several good sources for tissue-culture glassware.

b. Falcon Plastics, 5500 W. 83rd Street, Los

Angeles, Calif. 90045.

c. Brockway Glass, Inc., 11000 W, McNichols

Road, Detroit, Mich. 48221.

d. Becton, Dickinson and Company, Rutherford, N. J. 07070.

e. General Food Container Company, 1016

East 11th Street Indianapolis, Ind. 46202.

1 Labline Instruments, 3070-82 W. Grand

Avenue, Chicago, 111. 60622 and National

Appliance Company, 10855 S. W. Greenburg Road, P. 0. Box 23008, Portland, Oreg.

97223, are two of several incubator sources.

g. Saylor Beal, St. Johns, Mich. 48879.

h. Zeiss (W. H. Kessel and Company), 510 N.

Dearborn Street, Chicago, 111. 60410; Unitron Instrument Company, 66 Needham

Street, Newton, Highland, Mass. 02161.

i. Grand Island Biological Company, P. 0. Box

68, Grand Island, N. Y, 14072 ; Microbiological Associates, 4813 Bethesda Avenue, Bethesda, Md. 20014; and Hyland Laboratories,

4501 Colorado Boulevard, Los Angeles,

Calif. 90039, are three companies utilized

by the Regional Poultry Research Laboratory, USDA, East Lansing, Mich.

j. DIFCO, 920 Henry Street, Detroit, Mich.

48201.

k. Colorado Serum Company, 4950 York

Street, Denver, Colo. 80216.

1. Barnstead Company, 225 Rivermoor Street,

Boston, Mass. 02132. Mixed bed cartridge,

#0808.

m. Nutritional Biochemicals Corporation,

21010 Miles Avenue, Cleveland, Ohio 44128.

n. Johns-Manville Company, 22 E. 40th Street,

New York, N. Y. 10016.

0. Consolidated Laboratories, Incorporated, P.

0. Box 234, Chicago Heights, 111. 60412,

p. Technical Instrument Company, 348 Sixth

Street, San Francisco, Calif. 94103.

q. Flow Laboratories, 1206 Twinbrook Parkway, Rockville, Md. 20852.

r. Cooke Engineering Company, Medical Products Division, 735 North Street, Alexandria,

Va. 22314.

s. ROBOZ Surgical Instrument Company, 810

18th Street N.W., Washington, D, C. 20006.

Use of the Hemacytometer

A Spencer bright-line hemacytometer is

used. It is ruled into two areas of 9 mm.^ each.

These in turn are divided into nine squares,

each with an area of 1 mm." and each bounded

by a group of three lines. The central square

millimeter is the one used for counting cells. It

is ruled into 25 groups of 16 small squares, each

group separated by triple lines—the middle one

is the boundary. The ruled surface is 0.1 mm.

below the cover glass ; hence the volume of fluid

over 1 mm.^ is 0.1 cubic mm. The number of

cells in this 0.1 cubic mm. volume is then

counted and the value multiplied by 10,000 to

convert to cells per milliliter.

The procedure for counting is as follows : A

clean cover glass is placed onto the supporting

ribs of the counting chamber of a clean hemacytometer. The cell suspension to be counted

is dispersed and a small amount is removed

with a sterile capillary tube. The capillary is

placed on the counting chamber at the edge of

the cover glass. With this method, the fluid is

drawn rapidly into the chamber. Excess fluid

should be avoided. Either all 25 squares in the

middle 1 mm.'^ are counted and multiplied by

10,000 or the four corners and one middle

square are counted and multiplied by 50,000

(only 1/5 of the area counted). Cells on two

sides of the outer boundary are included in the

total count. Both sides of the hemacytometer

are counted and an average is determined. The

slide is cleaned, and the volume of cell suspension to add to the petri dishes to give the desired

cell number is calculated. Fresh medium is

added as needed to give the final volume of fluid

in the petri dish.

•Ù U.S. GOVERNMENT PRINTING OFFICE; 1971 0—415-963