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AFRICAN HORSESICKNESS VIRUS
THE

PLUM ISLAND
ANIMAL
DISEASE
CENTER

Research on
Foreign
Diseases of
Animals

AFRICAN SWINE FEVER VIRUS

SHEEP POX VIRUS

RINDERPEST VIRUS

U.S. DEPARTMENT
OF AGRICULTURE

AGRICULTURAL
RESEARCH SERVICE

CONTENTS

Page
Location and History 24 jyci.cits hres eee rece eee if
The: Center's Mission: .22.5o0se4 ons oe ee il
Safety Precautions: % s<. ¢...cbos there Rie ee eee 3
SUP pPOrt Activities) << 5.s.c aus eeme eel ees Bee Srna pears ey am emenars +)
Diseases Studied). nia2 eas ee a ee Oe ee 6
General Areas‘of Researches ens oer te ne Oe ee 7
Research Disciplines: 42.45.42 nea oo oe SO Oe oe 8
Research: Highlights). .2< acsucere ior cUsuenck a0 Aces ster Nees ete enn a eerie le
The Center’s Nugure> 5.5 acne eee eee Oe 13

Note: The pictures of the virus particles on the cover are electron
micrographs taken at Plum Island. They are magnified c. 175,000 times.

This publication supersedes Miscellaneous Publication 931, ‘‘The
Plum Island Animal Disease Laboratory.”

Washington, D.C. Issued May 1975

For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, D.C., 20402 - Price 40 cents
Stock Number 001-011-00163-1

THE PLUM ISLAND

ANIMAL DISEASE CENTER

Research on Foreign Diseases of Animals

The Plum Island Animal Disease Center is the
only research facility in the United States
devoted to the study of contagious foreign
diseases of animals. It is located on an island east
of Long Island, N.Y., and is operated by the
Agricultural Research Service, U.S. Department
of Agriculture.!

The Department is responsible for’

@ Developing capability of diagnosis of
animal diseases that do not exist in the United
States.

e Conducting basic and applied research on
foreign animal diseases and their causative organ-
isms.

e Developing adequate procedures so that
foreign, domesticated, and wild animals, and
semen, meat, and other animal products may be
imported safely.

The Center conducts fundamental research to
develop the necessary knowledge that enables
the Department to carry out these responsibili-
ties. The main objective is to prevent the
introduction of diseases that could result in high
death tolls or serious economic losses in our
susceptible livestock population.

LOCATION AND HISTORY

Plum Island is located 110 miles from New
York City, about 10 miles from Connecticut,
and about 1-1/4 miles off the northeastern end
of Long Island, N.Y. The island contains about
800 acres and is about 3 miles long and a mile
wide in the western, or widest part. It is reached
by boats operated by the Center from Orient

'Mailing address: P.O. Box 848, Greenport, Long
Island, N.Y. 11944.

Point, Long Island, where the Center has a
harbor, an office building, and storage facilities
for incoming supplies.

Plum Island was named by early explorers
who observed beach plums growing along the
shores. In 1659 the ruling Indian chief of Long
Island sold Plum Island to the first European
owner, Samuel Wyllis, for ‘‘a coat, a barrel of
biscuits, and 100 muxes’ or fishhooks.”’

The U.S. Government bought the island in the
1890’s and established Fort Terry, a coast
artillery post. The island was assigned to the
Army Chemical Corps after World War II. On
July 1, 1954, all of Plum Island, except for a
U.S. Coast Guard lighthouse, was formally trans-
ferred to the U.S. Department of Agriculture for
the establishment of a Center for the study of
exotic diseases of domestic animals.

Preliminary studies were started in 1954.
When additional laboratory facilities became
available in 1956 the Center’s research was
expanded into a broad program covering many
foreign animal diseases.

THE CENTER’S MISSION

The mission of the Center is to perform the
following basic and applied research and service
work on the various contagious foreign diseases
of animals, with primary emphasis on foot-and-
mouth disease.

Research

e Basic research on viral structure, patho-
genesis of the disease, antigen-antibody

2Muxes are small drills the Indians used to make holes
in wampum.

PN-3648

Aerial view of Plum Island.

reactions, and host and disease-agent relation-
ships.

e Applied research on virus survival in
animals and animal products, methods of virus
inactivation, and development of vaccines and
other control measures.

Service

e Diagnosis of disease by laboratory tests on
specimens from animals in suspected field out-
breaks.

e Tests for infectious agents in semen or
specimens from live animals prior to importa-
tion.

.@ Assessment of hazards from imported prod-
ucts.

e Production of diagnostic materials for
other laboratories.

e Training of U.S. and foreign personnel.

e Technical assistance to foreign countries to
lower disease rates and thus reduce hazards to
the United States.

Technical support to other Federal agencies
includes diagnostic services, specialized studies
on animal products, and development and
evaluation of new techniques.

Emergency services are performed as required
for diagnosing foreign animal diseases. When

2

materials from disease outbreaks of suspected
foreign origin are submitted to control agencies,
studies are conducted to determine whether a
foreign animal disease is involved.

Tests are made on throat fluids, serum, and
semen to determine whether animals or semen
for importation may be infected with foot-and-
mouth disease virus.

Training courses are given so that diagnosti-
cians in the field will become more familiar with
the foreign diseases of animals, which they may
have to recognize and investigate if outbreaks
occur.

Specialized studies on animal products such as
meat and semen are made to assist control
agencies in deciding whether certain animal
products should be admitted from foreign coun-
tries and what may be done to render them safe
from a disease standpoint. Certain foreign bio-
logical products require a similar safety evalua-
tion. If this service had been available in 1908,
an outbreak of foot-and-mouth disease in this
country might have been averted. The outbreak
was traced to contaminated imported smallpox
vaccine, which was propagated in calves.

New disinfectants and sterilization techniques
also are evaluated to assist the work of control
agencies in dealing with foreign animal diseases.

SAFETY PRECAUTIONS

Because of the Center’s location as well as its
special facilities, devastating foreign animal
diseases can be studied without endangering
livestock on the mainland. Congress provided
this protection for U.S. livestock by specifying
that the Center be on an island entirely under
Federal control and be separated from the
mainland by deep navigable water.

Rigid safety regulations were also devised to
prevent the escape of highly communicable
disease- causing agents from one research area to
another and the accidental introduction of
extraneous domestic disease agents, which

would complicate the studies.

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~~ ===—_

The Federal Government controls all move-
ment to, from, and on the island. Only author-
ized persons are permitted entry to the island;
entrance to the laboratory buildings and animal
quarantine areas is_ restricted. All Center
personnel are prohibited from contact with
susceptible species of animals, or premises where
such animals are held, for specified periods of
time after leaving the island (7 days for persons
working in the laboratories and 3 days for other
personnel).

The two main laboratory buildings on Plum
Island were specifically designed for research on
highly communicable’ diseases and_ are
considered among the safest in the world for
work on animal viruses. All entrances and exits

PN-3649

The ‘‘M. S. Shahan” is used to transport employees to and from Plum Island.

Le : PN-3650
A scientist uses the electron microscope.

for personnel, animals, and supplies are strictly
controlled. Persons must change to laboratory
clothing upon entering the building. Upon
leaving, they must take a decontaminating
shower before putting on their own clothing.
Exhaust air from these buildings is decontami-

\ em! = = nated through a system of filters, and all liquid

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x =
PN-3651 PN-3652
A technician inoculates chicken egg embryos to detect Foreign scientists observe a rapid diagnostic test for
virus. African swine fever.

————

PN-3653
A technician disinfects a truck that must be removed
from Plum Island.

wastes are sterilized by heat before being dis-
charged. Solid wastes, including animal
carcasses, are destroyed by incineration within
the research buildings.

SUPPORT ACTIVITIES

Because of its isolation, the Center maintains
all services needed to support its research. It
employs an administrative staff, engineers,
animal caretakers, maintenance men, a safety
staff, guards, firemen, and other workers, in
addition to scientists and laboratory technicians.

PN-3654
Cattle are introduced into the laboratory through air
locks.

PN-3655
Genetically stable laboratory mice are raised especially
for Plum Island research.

The Office of the Director provides overall
guidance and management for the research and
supporting groups.

Administrative Management Services is
responsible for personnel work, purchase and
delivery of supplies, and operation of food,
photographic, and duplicating services.

The Safety Office places major emphasis on
preventing the escape of disease agents from the
Center. Other programs include industrial and
fire safety, first aid, and plant security.

Animal Supply maintains colonies of disease-
free guinea pigs, mice, and other small labora-
tory species to supply research sections. All large
experimental animals, such as cattle, sheep,
goats, and swine, brought to the island are
inspected. Quarantines are placed on all animals
until they are needed for research. Animal
Supply also provides whole blood, tissues, and
serum from normal animals for use in diagnostic
tests and tissue cultures.

Laboratory Services provide tissue cultures
and prepare media and sterile equipment for use
in the laboratory. This group also operates a
laundry and a glassware-washing service.

The Library acquires and makes available
scientific books, journals, and reports necessary
for animal disease research. It provides reference
and reprint services.

Engineering and Plant Management is
responsible for the maintenance, repair and
construction of laboratory facilities and
equipment, utility plants and systems, harbors
and docks, pavements and grounds and various
support structures. It also provides all utility

5

PN-3656

A supervisory data center is used to control and monitor equipment at Plum Island.

support services
heating, water,
processing plus
transportation.

such as electrical power,
sewage decontamination and
marine and _ automotive

DISEASES STUDIED

The contagious foreign animal diseases
studied and diagnosed in the Plum Island Center
and the principal domestic animals they infect
include—

e Foot-and-mouth disease—cattle, hogs, sheep,
goats.

e Rinderpest—cattle.

Teschen disease—hogs.

African swine fever—hogs.

Fowl plague—poultry.

African horsesickness—horses, mules, asses.
Asiatic Newcastle disease—poultry.

Lumpy skin disease—cattle.

Ephemeral fever—cattle.

goats, swine, horses.

Duck virus enteritis—ducks.

Vesicular exanthema of swine—swine.
Louping ill—sheep.

Ovine and caprine pox—sheep, goats.
Nairobi sheep disease—sheep.

Rift valley fever—sheep, cattle, goats.
Bovine herpes mammillitis—cattle.

Exotic vesicular stomatitis—cattle, sheep,

Swine vesicular disease—swine.

Borna disease—horses.

Peste des petits raminants—sheep, goats.
Equine encephalosis—horses.

Contagious bovine pleuropneumonia—

cattle.

Contagious caprine pleuropneumonia—

goats, sheep. v

Contagious agalactia—sheep, goats.
East Coast fever—cattle.

The diseases listed are caused by viruses
except for the last four. Contagious bovine and

caprine pleuropneumonias and contagious agal-
actia are caused by mycoplasmas and East Coast
fever by a blood parasite (hematozoan). Some of
the diseases affect wild animals and birds in
addition to domestic animals. About 70 percent
of the research and service work is devoted to
foot-and-mouth disease because of its great
economic importance. Techniques and materials
are being developed for rapid diagnosis of this
and the other foreign diseases in the event of
outbreaks here.

The Center’s program is flexible enough to
allow the study of additional disease problems
when necessary. An outbreak of duck virus
enteritis (duck plague), in the Long Island duck
industry, necessitated a comprehensive study of
this disease. When hog cholera is officially
eradicated from this country, it also will be
added to the list of foreign diseases to be studied
and diagnosed.

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GENERAL AREAS OF RESEARCH

Research at the Center has been divided into
five general areas. These areas broadly corres-
pond to the work of five different research
groups, or disciplines. The general areas of
research are as follows.

Viruses

(1) Biophysics

(2) Protein coat structure
(3) Nucleic acid structure
(4) Synthesis

(5) Antiviral agents
Vaccines

(1) Virus production

(2) Inactivants

(3) Adjuvants

(4) Safety and serologic testing
(5) Immunity challenge

SS
=
PN-3657

Standby electric generators are always ready in case power from Long Island is cut off.

Cell Cultures

(1) Kinds of cells

(2) Cell nutrients

(3) Virus yield

(4) Viral changes

(5) Interference

Control Measures

(1) Virus persistence in animals, products, and
environment

(2) Carrier studies

(3) Pathology and diagnosis

(4) Epizootiology and host range

(5) Disinfection

Diagnostic Tests

(1) Complement fixation

(2) Neutralization

(3) Agar gel diffusion

(4) Fluorescent antibody

(5) Agglutination

RESEARCH DISCIPLINES

The five research disciplines are biochemical
and biophysical, immunological, cytological,
microbiological, and diagnostic investigations.
These research disciplines are composed of
veterinarians, virologists, bacteriologists, patho-

logists, chemists, physicists, and their technical
assistants. Working alone or as teams, the
scientists and their assistants are assigned to one
of the five research disciplines.

Biochemical and Physical

Scientists in biochemical and physical investi-
gations are concerned with problems in mole-
cular biology. These scientists produce milligram
quantities of foot-and-mouth disease virus in
cultures of baby hamster kidney cells and purify
the virus for use in biochemical and immunolo-
gical studies. They examine animal virus
particles for their size and shape by electronmic-
roscopy and for their chemical properties,
including resistance to mechanical treatment, pH
changes, thermal changes, and variations in ionic
strength. They determine the effects of enzymes
and chemicals as purifying agents and inacti-
vants; study viruses intact and broken down into
their protein and infectious nucleic acid subu-
nits; determine diffusion, electrophoretic, and
sedimentation rates of viruses and their sub-
particles.

The scientists in this group also investigate the
correlations of physiochemical properties with
infectivity, immunogenicity, and antibody-

PN-3658

An electron micrograph shows foot-and-mouth disease virus particles.

antigen relationships. They study the mechanism
of virus synthesis and its inhibition in tissue
culture and cell-free systems by biological and
chemical methods, using radiobiological tracers.

Immunological

Scientists in immunological investigations
check the response of animals infected with or
vaccinated against disease agents and they con-
duct research on the antibodies that protect
against disease. Serum from these animals is
separated into elemental components, and these
are then analyzed by serological, chemical, and
animal-testing techniques. They also study anti-
gens or viruses that cause the disease.

Another function of this group is the develop-
ment and testing of vaccines appropriate for
possible use if established disease-eradication
procedures should fail to control invasions of
foreign diseases. These studies include chemical
inactivation of virus and development of critical
tests to determine the safety and potency of
vaccines produced. Such research requires the

: PN-3659
A technician operates a shadow casting instrument so that virus preparations can be viewed as three-dimensional objects.

vaccination of many animals and the study of
their immunity by serological and challenge
methods.

Cytological

Scientists in cytological investigations study
viral inhibitory substances, changes in viruses
caused by environmental conditions, and growth
of viruses in cell cultures. They have found that
treatment of cells with certain chemicals stimu-
lates the production of inhibitory substances
effective to a limited extent against foot-and-
mouth disease virus. They are investigating the
possibility that these substances may be useful
in the prevention of the disease.

Other scientists are attempting to change the
foot-and-mouth disease virus by various proce-
dures so that it no longer produces disease and
thus might be used as a live attenuated virus
vaccine.

Work in the cytological group also includes
investigations on the viral susceptibility of
various types of cell cultures and factors that

9

m2

PN-3660

A scientist studies the results of foot-and-mouth disease virus on a ratio recording instrument.

affect their susceptibility. These studies are
being done to obtain highly susceptible cultures
for diagnosis of viral diseases and for other work
involving assay or production of viruses.

Microbiological

Scientists in microbiological investigations
study the susceptibility of various species of
animals to virus diseases, explore ways in which
the diseases spread, and determine in what
organs and tissues the virus may be found. They
study the factors that result in animals becoming
virus carriers. They also trace the survival of
viruses in meat, blood, semen, and other animal
products. From the results of these studies, the
U.S. Department of Agriculture is able to assess
the hazards of importing live animals and animal

10

materials from foreign countries in which
dangerous diseases exist.

Scientists also study the effects of chemical
and physical environments on viruses and thus
contribute to knowledge regarding methods of
virus inactivation, disinfection of contaminated
materials and premises, and survival of viruses
under various conditions. Such information is
vital in preventing disease and eradicating out-
breaks.

Diagnostic

When USDA veterinary diagnosticians in the
field observe animals showing clinical signs
suspicious of foreign animal disease, they collect
samples and submit them to Plum Island. At

Plum Island, the staff of diagnostic investiga-
tions conducts varicus serological tests, virus
isolations, animal inoculations, and pathological
studies to determine if the samples were positive
or negative for a foreign animal disease.

Considerable work is required to have in
readiness all of the virus strains, antiserums, and
cell cultures needed for the various diagnostic
tests. In addition, research is conducted on
various aspects of foreign diseases of animals
other than foot-and-mouth disease.

RESEARCH HIGHLIGHTS

Foot-and-Mouth Disease (FMD)

Viewed foot-and-mouth disease virus (FMDV)
in the electron microscope as a spherical particle
23 nanometers (about one-millionth inch) in
diameter with 32 capsomeres on its surface.

Established that FMDV ribonucleic acid:

(RNA) can be encapsidated in a bovine entero-
virus (BEV) protein coat and that such viral
particles have biophysical properties of BEV but
can produce FMDV in further growth cycles.

Developed an in vitro system for the study of
the synthesis of FMDV-RNA.

Showed that FMDV-infected cells contain an
enzyme, RNA polymerase, which is induced by

PN-3661

A technician cultures swine blood to determine
sterility.

PN-3662
A technician dispenses kidney cells into tissue-culture
bottles.

the virus, is necessary for viral replication, but is
not found in normal cells.

Showed that the virus replication activity of
RNA polymerase is inhibited by antibodies
produced in FMD-infected animals. Thus, RNA
polymerase may be identical to a virus-infection-
associated antigen (VIA).

Demonstrated that VIA made from extracts
of FMDV-infected cell cultures reacted with
antibodies in serums from FMD-infected animals
in agar gel precipitin tests, forming a band
distinct from that of whole FMDV. A similar
finding was demonstrated by means of fluores-
cent antibody techniques.

Established that the FMDV infection
associated antigen used in the VIA agar gel
diffusion test was a valuable tool for epizootio-
logical surveys.

Found that FMDV persisted in cell cultures
made from the pharyngeal and esophageal cells
of infected cattle for as long as 24 weeks.

Found that FMDV can infect cattle, sheep,
and goats and multiply in the upper respiratory
tract regardless of their immune status, and that
this infection and multiplication can occur in
the complete absence of clinical signs.

Showed that a steer after intranasal inocula-
tion with FMDV could transmit FMDV for 7 to

i

PN-3663

A technician shows plaques formed by virus particles found in the blood of steers.

8 days and that the most infectious period was
during the third day.

Found a latent form of FMD characterized by
virus isolation from the blood in the absence of
specific antibody development and_ with
extremely long incubation periods.

Determined that FMDV may be present in
semen of infected bulls before and after clinical
signs of disease and that it may be transmitted
to cows by artificial insemination.

Established that FMDV survives in lymph
nodes and blood of beef carcasses for as long as
60 days, in bone marrow for more than 6
months, and in lymph nodes of wet, salt-cured
meat for as long as 50 days.

Developed radial immunodiffusion procedures
for measuring FMDV in crude tissue culture
fluids as well as in concentrated and purified
preparations.

Showed that FMDV could be spread by air
from infected to clean areas.

Established a facility for the large-scale culti-
vation of baby hamster kidney cells in rolling

12

bottles and the production, therefrom, of 100
milligrams per week of purified FMDV.

Determined the dose of ionizing radiation
required to inactivate FMDV and its RNA.

Found that FMDV is inactivated by organic
acids, and by ethylene oxide gas, when sufficient
humidity is present, and _ that _beta-
propiolactone, acetylethyleneimine, ethylene
oxide may be used as inactivants when retention
of antigenicity is desired.

Used polyethylene glycol for precipitation of
FMDV for vaccine production.

Showed that a FMDV vaccine combining oil
adjuvant can be used in a vaccination program
involving revaccination, reducing the number of
vaccinations per year and giving adequate pro-
tection for 6 months or longer.

Determined that purified FMD viruses inacti-
vated.and combined with oil adjuvants produced
the first satisfactory vaccine for swine.

Showed the possible relationship between
swine vesicular disease virus and Coxsackié B 5
virus of humans.

PN-3664

Plaques formed by foot-and-mouth disease virus particles
in tissue culture cells.

Other Diseases

Isolated vesicular stomatitis virus from an
infected human being.

Developed a rapid laboratory diagnostic test
for African swine fever, in cooperation with the
East African Veterinary Research Organization.

In cooporation with the same group, estab-
lished methods for growing the schizont form of
East Coast fever parasites in cell cultures. This
technique modifies the parasite for potential use
in a vaccine.

Developed, in cooperation with the East
African Veterinary Research Organization, diag-
nostic tests for contagious bovine pleuro-
pneumonia in formalized lung tissues using
fluorescent antibody and agar gel diffusion
techniques.

Purified the attenuated duck enteritis virus
and developed a seed virus, which was supplied
to the duck industry for production and use as a

vaccine. This work was made possible by co-
operation of Dutch scientists who supplied the
starting materials.

THE CENTER’S FUTURE

As the world human population increases and
the food supply becomes less abundant for each
individual, the need to reduce losses from animal
diseases becomes more important. The Center
already has found and will continue to find new
ways such as rapid diagnostic tests, control
measures, and vaccines to limit or prevent
outbreaks of foreign animal diseases.

Increased demand for food supplies involves
developing faster growing and improved types of
livestock, which in turn requires importation of
animals and semen with the special genetic
background to develop inbred and hybrid pro-
geny of the desired type. Here again the Center
is called upon to develop sensitive tests for
detecting disease agents so that such importa-
tions may be made with a minimum of risk.
Thus the Center has an important role in the
development of future food supplies from live-
stock.

Basic research at the Center should continue
to develop new techniques and concepts. How-
ever, applied research will receive more emphasis
than it has in the past to put into service the
improved techniques and findings that have been
made through basic studies. Also, the results will
have application to many other branches of
medical science.

The very nature of research prevents the
prediction of the exact character and the timing
of conclusive results, but achievements of the
program already have been outstanding. In the
years ahead, the Plum Island Animal Disease
Center undoubtedly will continue to add to the
achievements of U.S. and international research.

13

Prepared by

The Plum Island Animal Disease Center,
Northeastern Region, Agricultural Research Service

U.S. GOVERNMENT PRINTING OFFICE: 1975 O—576—530