AN ABSTRACT OF THE THESIS OF
JAMES R. WANGELIN
for the degree of MASTER OF SCIENCE
in VETERINARY MEDICINE presented on
December 14, 1978
Title: VACCINATION STUDIES WITH BOVINE ADENOVIRUS
TYPE 3 IN A DAIRY CALF HERD
Abstract approved:
Redacted for privacy
Donald E. Mattson
In October 1977, a study was undertaken to determine the role
of bovine adenovirus type 3 in enzootic pneumonia of calves, and the
efficacy of using a vaccine for prevention of disease. Bovine adenovirus type 3 was isolated from the herd under study in the previous
year. Calves in the herd were experiencing enzootic pneumonia
associated with conjunctivitis and keratoconjunctivitis. Initial signs of
disease included excessive lacrimation, pyrexia, nasal discharge and
coughing. As the disease progressed, effected animals exhibited
respiratory embarassment (especially upon exertion), malaise, low
weight gains, and pneumonitis (as determined by the presence of
dyspnea, and by stethoscopic examination for the detection of abnormal
lung sounds).
Forty-two one to five day old calves were randomly chosen for
the study. Twenty-one calves were vaccinated with bovine adenovirus
type 3 experimental vaccine. The vaccine consisted of a killed viral
preparation with an aluminum hydroxide adjuvant, and was prepared
by a commercial biological firm. The remaining twenty-one calves
were unvaccinated controls.
Two of the vaccinated calves experienced pneumonia, nineconjunctivitis and two--keratoconjunctivitis. No fatalities occurred
among the vaccinated calves. Fourteen of the vaccinated calves pro-
duced a four-fold or greater rise in the titer of serum-viral neutralizing antibodies to bovine adenovirus type 3, strain 5C. Five calves had
high levels of colostral viral serum-neutralizing antibodies which
interferred with active antibody production. Two calves had low
levels of colostral antibodies and low levels of active antibody production. No adverse side-effect was noted at the vaccination site in any
of the vaccinated animals.
Eleven of the control calves experienced pneumonia, three- -
conjunctivitis and elevenkeratoconjunctivitis. Eight of the control
calves died. Seven of the control calves produced a four-fold or
greater rise in the serum-viral neutralizing antibody titer to bovine
adenovirus type 3, strain 5C.
Bovine adenovirus type 3 was isolated from five of the 42 calves.
A second virus, believed to be bovine papular stomatitis virus, was
isolated from seven calves. In only one case were both viruses
isolated from the same calf.
Vaccination Studies with Bovine Adenovirus
Type 3 in a Dairy Calf Herd
by
James Richard Wangelin
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed December 1978
Commencement June 1979
APPROVED:
Redacted for privacy
Associate.--1)rofessor of Veterinary Medicine
in charge of major
Redacted for privacy
Dean, School of Veterinary Medicine
Redacted for privacy
Dean of Graduate School
Date thesis is presented
December 14, 1978
Typed by Clover Redfern for
James R. Wangelin
ACKNOWLEDGMENTS
I would like to thank Dr. Mattson for his patience and understanding throughout all phases of this project. I thank Ms. Patsy
Smith for her technical assistance and Mr. Brian Grinnell for his
assistance in preparing the photographs. I thank my parents and
family for their interest and encouragement.
TABLE OF CONTENTS
Page
Chapter
I.
II.
III.
INTRODUCTION
1
LITERATURE REVIEW
3
Clinical Diseases Associated with Adenoviral Infection
Human Diseases Associated with Adenoviral
Infection
Diseases of Other Animal Species Associated
with Adenoviral Infection
Classification of Bovine Adenoviruses
Natural and Acquired Immunity Against Bovine
Adenoviral Infection
Classification of the Family Adenoviridae
Characteristics of Adenoviruses
3
MATERIALS AND METHODS
Media
Cell Cultures
Primary Trypsinization
Primary Culture Techniques
Secondary Trypsinization
Vaccination and Sampling
Samples Taken
Inoculation in Cell Culture
Viral Pool Preparation
Viral Identification
Cytopathic Effect and Inclusion Body
Characteristics
Chloroform Sensitivity
Serum-Viral Neutralization Tests
Viral Ultrastructure
Inoculation into Embryonated Chicken Eggs
Challenge
Viral Isolation Attempt from the Experimental Vaccine
Serum-Viral Neutralization Titer
IV. RESULTS
Clinical Observations
Viral Isolation
Viral Characterization: Bovine Adenoviruses
Behavior in Cell Cultures
4
5
10
12
14
15
20
20
21
21
22
23
23
24
26
26
27
27
28
28
29
30
30
31
31
32
32
32
44
44
Chapter
Viral Titration and Serum-Viral Neutralization
Tests
Electron Microscopy
Viral Characterization: Bovine Papular Stomatitis Virus
Behavior in Cell Cultures
Chloroform Sensitivity
Replication in Cells and the Chorioallantoic
Membrane
Electron Microscopy
Serologic Response to Vaccination and/or Natural
Infection
Page
45
45
45
45
46
47
47
47
Clinical Observations
Response to Vaccination
65
65
68
BIBLIOGRAPHY
71
V. DISCUSSION
LIST OF FIGURES.
Fig ure_
1.
2.
3.
4.
5.
Page
Photomicrograph of cytopathic effect observed in bovine
embryonic kidney cell cultures 4-5 days post inoculation
with bovine adenovirus type 3 isolate R 87 C/N.
51
Photomicrograph of a normal bovine embryonic kidney
cell culture.
51
Photomicrograph of a mature intranuclear inclusion body
observed in hematoxylin and eosin stained bovine
embryonic kidney cell culture 5 days after inoculation
with bovine adenovirus type 3 isolate R 87 C/N.
53
Photomicrograph of a mature intranuclear inclusion body
observed in bovine embryonic kidney cell culture stained
with acridine orange 5 days after inoculation with bovine
adenovirus type 3 isolate R 87 C/N.
53
Photomicrograph of a normal bovine embryonic kidney
cell culture stained with acridine orange.
55
6.
Electron micrograph of three negatively stained bovine
adenovirus type 3 virions.
7.
Electron micrograph of a large group of negatively stained
bovine adenovirus type 3 virions.
57
Photomicrograph of early cytopathic effect observed in
bovine testicular cell cultures 2-3 days after inoculation
with bovine papular stomatitis virus isolate R 88 C /N.
57
Photomicrograph of later cytopathic effect observed in
bovine testicular cell cultures 8 days after inoculation
with bovine papular stomatitis virus isolate R 88 C /N.
59
Photomicrograph of a normal bovine testicular cell
culture.
59
Photomicrograph of a horse-shoe shaped intracytoplasmic inclusion body observed in an acridine orange
stained bovine testicular cell culture 7 days after inoculation with bovine papular stomatitis virus isolate
R 85 C/N.
61
8.
9.
10.
11.
Figure
12.
13.
14.
Page
Photomicrograph of a large intracytoplasmic inclusion
body observed in acridine orange stained bovine
testicular cell culture 7 days after inoculation with
bovine papular stomatitis virus isolate R 85 C/N.
61
Photomicrograph of a normal acridine orange stained
bovine testicular cell culture.
63
Electron micrograph of two negatively stained bovine
papular stomatitis virions.
63
LIST OF TABLES
page
Table
I.
II.
Clinical history of vaccinated calves during the
13-week observation period.
33
Clinical history of control calves during the 13-week
observation period.
38
III. Bovine adenovirus type 3 and bovine papular stomatitis
IV.
V.
virus isolation results.
43
Viral-serum neutralization titer against bovine
adnovirus type 3, strain 5C: vaccinated calves.
49
Viral-serum neutralization titer against bovine
adenovirus type 3, strain 5C: control calves.
50
VACCINATION STUDIES WITH BOVINE ADENOVIRUS
TYPE 3 IN A DAIRY CALF HERD
I. INTRODUCTION
Respiratory disease is the leading disease problem in cattle in
the United States (McKercher, 1968). A wide variety of respiratory
infections exist, collectively referred to as the bovine respiratory
disease complex. The respiratory disease complex includes acute
inflammatory reactions within the nose, paranasal sinuses, larynx,
trachea, bronchi and lungs. Conjunctival infection may occur simultaneously with respiratory infection. Many etiologic agents are
involved in the complex. Although the respiratory system is the
initial anatomic location of many of the infections in the respiratory
disease complex, pathologic changes resulting from viremia or
septicemia may occur in organ systems outside the respiratory system (Jensen, 1968).
Pneumonias are the most frequently occurring respiratory
infections in calves. These penumonias have been referred to as calf
influenzal pneumonia, pneumoenteritis, enzootic pneumonia, calf
pneumonia-enterities, inclusion body pneumonia and cuffing
pneumonia (McKercher, 1968). Clinical signs may vary with each
pneumonia type, and some types are seen more often in different ages
of calves (Mattson, personal communication).
2
Respiratory infections of cattle may arbitrarily be grouped into
three categories
(1) infections in which a virus is the sole etiologic
agent; (2) infections initiated by a virus or viruses, but in which
secondary bacterial infection is largely responsible for the severity
of the condition and the resulting pathologic damage; and (3) infec-
tions in which one or more species of bacteria are believed to be the
sole etiologic agents. Most respiratory infections fall into the second category (McKercher, 1968).
Environmental factors play an extremely important role in the
epizootiology of respiratory infections (Jensen, 1968).
The current investigation was made to determine the etiologic
role of bovine adenovirus type 3 in an outbreak of enzootic pneumonia,
and the efficacy of vaccinating one to five day old calves parenterally
with a formalin killed bovine adenovirus type 3 monovalent vaccine,
in an attempt to lessen or alleviate lower respiratory tract disease.
3
II.
LITERATURE REVIEW
Adenoviruses were first recognized in 1953, when cultures of
human adenoid tissue were observed to undergo characteristic degeneration. The adenoid-degenerating agent isolated from these cultures
was shown to be transmissible in tissue cultures and possessed characteristics of a virus (Rowe et al. , 1953). Following this initial
discovery, adenoid-degenerating agents were isolated from nasopharyngeal and conjunctival secretions and feces of patients with
acute and undifferentiated respiratory disease (Hilleman et al. , 1954,
and Rowe et aL , 1955). In 1954, the term radenoidal-pharyngeal-
conjunctival agents° was used as the generic designation for this new
group of viruses (Huebner et al., 1954). In 1956, the group name was
changed to adenoviruses (Wildy, 1971).
Clinical Diseases Associated with Adenoviral Infection
Adenoviruses are responsible for a number of diseases of
human and veterinary medical significance. In human infections
adenoviruses are mainly involved in the etiology of acute respiratory
illnesses (Medical Research Council, 1965), but when natural hosts of
all species are considered, the respiratory, digestive and lymphatic
systems are most commonly affected. Conjunctivitis may accompany
respiratory infection. In certain hosts hepatitis is common. Infection
4
of the urinary tract occurs less frequently (Pereira, H.G. , 1975).
Human Diseases Associated with Adenoviral Infection
Clinical disease signs resulting from human adenoviral infection
generally include pyrexia, pharyngitis, tonsillitis and cough (Pereira,
M.S., 1973). Adenoviruses have been isolated from clinical cases of
pharyngitis-bronchitis, mild upper respiratory disease (Brandt et al.,
1969), pertussis-like illness (Connor, 1970; Olson, 1950; and Pereira,
M.S., 1971), sporadic and epidemic pneumonia (Chany et al., 1958;
Jen et al. , 1962; Teng, 1960; and Van der Veen, 1962), bronchiolitis
obliterans and bronchioectasis in children (Becroft, 1971), epidemic
keratoconjunctivitis (Pereira, H. G. , 1975), conjunctivitis (Beale
et al., 1957) and hemorrhagic cystitis (Mufson et al., 1971 and 1973).
Other human diseases more remotely associated with adenoviral
infection are hepatitis (Alterman et al. , 1973 and Hatch et al. , 1966),
roseola infantum (Neva et al., 1954) and other exanthamous diseases
(Kjellen et al. , 1957 and Mufson et al. , 1971), acute thyroiditis
(Swann, 1964), thymic dysplasia (Alterman et al., 1973), ocular-
urethro-synovial syndrome (Thabaut et al., 1962) and meningoen-
cephalitic infections (Enjalbert et al., 1962; Falukner et al. ,
1962;
and Gabrielson et al., 1972).
Human adenoviruses may infect rabbits (Pereira, H. G. et al.,
1957), dogs (Siena et al., 1960) and calves (Bettinotti, 1966) under
5
experimental conditions, and produce pneumonia in colostrum-
deprived newborn pigs (Betts et al., 1962) and fatal infections of
hamsters and mice (Fenner, 1976).
Diseases of Other Animal Species Associated with
Adenoviral Infection
Avian adenoviruses are widely distributed and have been
isolated from the peripheral blood, bursa of Fabricius, central
nervous system, feces, conjunctiva, pharynx, lungs, liver and
spleen of diseased fowl (Aghakhan, 1974). Avian adenoviral infection
has been associated with central nervous disease, inclusion body
hepatitis, quail bronchitis, air sacculitis, hemorrhagic enteritis and
marble spleen disease (Aghakhan, 1974 and Mc Ferran, 1977).
Experimentally it has been shown that avian adenoviruses are coetiologic agents with other pathogens (Pereira, H. G. , 1975).
Canine adenovirus type 1 is the etiologic agent of infectious
canine hepatitis (Kapsenberg, 1959), while canine adenovirus type 2
has been shown to cause laryngotracheitis (kennel cough) (Ditchfield
et al. , 1962).
Murine adenoviruses were first isolated from apparently
normal mice. Natural infections with murine adenoviruses are gen-
erally subclinical, although the viruses localize in the kidney and are
excreted in the urine. Experimental infection with murine
6
adenoviruses may cause fatal disease in suckling mice with lesions in
brown fat, myocardium, adrenal cortex, salivary glands and kidneys
(Hartley et al.
,
1960 and Heck et al. , 1972). Persistent renal infec-
tion has been shown to predispose mice to acute pyelonephritis
(cinder, 1964).
Equine adenoviruses have been isolated from horses with
respiratory diseases in the United States, Canada, Australia and
Germany (McChesney et al., 1977) and from cases of pneumonia in
foals with combined immunologic deficiency (McChesney et al., 1973).
Ovine adenoviruses have not been directly associated with overt
disease, but there has been an increased incidence of pneumonia in
flocks where adenoviruses were isolated (McFerran et al., 1971).
Simian adenoviruses have most frequently been isolated from
cultures of monkey kidney cells which have been used for the propaga-
tion of other viruses (Hull, 1968). Several simian adenoviral types
have been isolated from cases of respiratory and enteric infections
and conjunctivitis (Bullock, 1965 and Pereira, M.S. , 1975). Adenoviruses, antigenically related to, but distinguished from human adeno-
viruses, have been isolated from chimpanzees with hepatitis (Hillis
et al., 1968 and 1969).
Porcine adenoviruses have been isolated from pigs with interstitial pneumonia and myocarditis, non-suppurative meningoencephalitis (Moffatt et al. , 1974 ), diarrhea (Derbyshire et al., 1966 and
7
Haig et al., 1964) and pneumonia (Mc Ferran, 1975).
Bovine adenoviruses have been isolated from apparently healthy
cattle, and cattle with clinical signs of pneumonia (Cole, 1971 and
Phillip, 1970), pneumoenteritis (Aldasy et al., 1965; Bartha et al.,
1964 and 1966; Darbyshire et al., 1965; and Mattson, 1973), respira-
tory tract disease (Darbyshire et al., 1965; Ide, 1970; and Rondhuis,
1970 and 1975), conjunctivitis and keratoconjunctivitis (Wilcox, 1969),
febrile respiratory disease associated with rhinorrhea and diarrhea
(Tanaka et al., 1968), and from stillborn or aborted calves, and
calves suffering from weak calf syndrome (Dierks et al. , 1977). Sev-
eral isolations of bovine adenoviruses have been made as latent
viruses in testicular cell cultures (Pereira, M.S. , 1975; Phillip
et al., 1972; and Rondhuis, 1968).
Bovine adenoviruses were first recognized in 1959 when two
prototypes were isolated in the United States from the feces of apparently healthy cattle (Klein et al., 1959 and 1960). In the summer of
1962, two simultaneous outbreaks of virus diarrhea occurred in
Miskole, Hungary. Initially viral isolation studies were directed to
confirm the presence of viral diarrhea virus, but isolation of this
virus failed. Upon further investigation adenoviruses were recovered.
Two strains were recovered from calves which had died of the disease,
and a third strain was recovered from the nasal secretions of a
diseased calf. In contrast to the two earlier prototype strains, these
8
three Hungarian isolates would only replicate in testicular cell
cultures. The three isolates were found to be identical with each
other but different from the bovine adenoviruses isolated previously
(Bartha et al., 1964). The following year another adenovirus type was
isolated from calves with the same disease syndrome (Aldasy et al.,
1965),
These isolates were later classified as bovine adenoviruses
types 4 and 5 (Bartha et al. , 1966).
Bovine adenovirus type 8 was also isolated from calves with
pneumoenteritis. Bovine adenovirus type 8, along with bovine
adenoviruses types 4 and 5 are believed to be the major etiologic
agents of pneumoenteritis in Hungarian calves (Bartha et al., 1970).
Bovine adenovirus type 3, strain WBR 1, was isolated from a
conjunctival swab from an apparently healthy cow in 1965 (Darbyshire
et al., 1965). However in later calf inoculation studies it was
demonstrated that WBR 1 was capable of producing pneumonia
(Darbyshire et al.
1966).
Although the clinical signs were generally
mild to moderate, two of six calves inoculated with bovine adenovirus
type 3, strain WBR 1, showed respiratory embarassment, and two
others later died. Calves infected experimentally with strains of
bovine adenoviruses types 1 and 2 developed mild respiratory disease
and histologic evidence of adenovirus pneumonia (Ide et al., 1969 and
Mohanty et al., 1965). This evidence suggests that bovine adenovirus
type 3 is the more significant pathogen of the first three types
9
(Darbyshire et al. , 1965).
Colostrum-deprived calves have been inoculated with strains of
bovine adenoviruses types 4 and 5 isolated in Hungary. These calves
developed penumonia and diarrhea, although older calves inoculated
with the same isolates showed no signs of disease (Aldasy et al.,
1965).
In other experimental animal studies, calves were inoculated by
a variety of routes with a Japanese strain of bovine adenovirus type 4.
The calves produced neutralizing and complement-fixing antibodies,
and exhibited a mild clinical response consisting of diarrhea, nasal
discharge, pyrexia, leukopenia and viremia of low titer and short
duration (Tanaka et al., 1968).
Colostrum-deprived calves inoculated with an American strain
of bovine adenovirus type 4 experienced pyrexia, soft feces, diarrhea
and viremia (Mattson et al. , article in press).
Two subgroup II bovine adenoviruses (types not determined),
which were isolated from stillborn or aborted calves, or calves suffering from weak calf syndrome were inoculated into healthy calves.
The experimentally infected calves developed a 2-3°C rise in tempera-
ture, nasal discharge and diarrhea on the third day post-inoculation.
By the seventh day post-inoculation no disease signs were evident
(Dierks et al., 1977).
10
Experimental infection of susceptible calves with both typed
and untyped bovine adenovirus strains generally show these viruses
to be pathogens, producing a mild clinical response characterized by
pyrexia, anorexia, respiratory distress, nasal and conjunctival discharge and occasionally diarrhea. In some cases a viremia is present
(Aldasy et al.
,
1965; Burki, 1973; Cole, 1971; Darbyshire, 1965;
Mattson, 1973b; Mayr et al. , 1970; Mohanty et al. , 1965; Rondhuis,
1970; Sickira et ad. , 1972; and Tanaka et al. , 1968).
The clinical
response of calves to experimental adenovirus infection suggests that
pneumonia is more severe if viral, bacterial or mycoplasmal coinfection occurs (Collier et al. , 1960; Phillip et al. , 1968; and Trapp
et al.,
1966).
Gross pathologic changes produced by adenoviral infection are
generally confined to the lungs and consist of interstitial pneumonia,
peribronchial reactions, and in some cases proliferative bronchitis,
necrosis, bronchiolar occlusion and alveolar collapse (Burki, 1973;
Cole, 1971; Darbyshire, 1965; Mattson, 1973b; Mohanty et al., 1965;
and Rondhuis, 1970).
Classification of Bovine Adenoviruses
Presently there are eight accepted types of bovine adenoviruses
(Rondhuis, 1975). Whereas adenoviruses of other animals and human
origin do not have clearcut distinctions between their various types
11
(other than serologic differences), the bovine adenoviruses are
heterogeneous in certain physical, chemical and biologic characteris-
tics. Accordingly, bovine adenoviruses are divided into two subgroups based upon these differences (Bartha, 1969).
The subgroup 1 bovine adenoviruses are represented by types
1-3.
The members of this subgroup: (a) share the soluble
complement-fixing antigen present in all human adenovirus types
(Bartha, 1969), (b) replicate in fetal bovine kidney cell cultures
(Mohanty, 1971),
(c) induce the formation of single intranuclear
inclusion bodies which are irregular in shape, (d) are more easily
isolated in early passages in cell culture, and (e) occasionally replicate in kidney cell cultures of other animal species (Bartha, 1969).
In addition to these differences, the subgroup I bovine adenoviruses
replicate primarily in the mucous membrane cells of the respiratory
and intestinal tract, even in the presence of circulating humoral anti-
body. Seldom is there a viremia (Mattson, article irLpress).
The subgroup II bovine adenoviruses are represented by types
4-8. Members of this subgroups (a) do not possess the complementfixing antigen which is present in human adenoviruses and subgroup I
bovine adenoviruses,
(b) replicate well in testicular or lung cell cul-
tures, (c) form many circular, sharply defined intranuclear inclusions, and (d) require several blind passages in cell culture for
isolation (Bartha, 1969). The subgroup II bovine adenoviruses are
12
thought to replicate in the vascular endothelial cells, or possibly in
the pharyngeal cavity or intestinal tract. Infection is accompanied by
viremia. During the viremic stage the virus may localize in the
respiratory and enteric tracts, or produce a chronic or latent infection in the gonads. Humoral antibodies may effectively inhibit virus
dissemination (Mattson, article in press).
Natural and Acquired Immunity Against
Bovine Adenoviral Infection
In experimentally infected newborn calves neutralizing anti-
bodies appear 5-6 days after virus inoculation, and reach a maximum
value 2-3 weeks post-inoculation (Cole, 1971 and Tanaka et al. , 1968).
Humoral antibodies are transferred to the calf through colostrum, but
are lost after 10-12 weeks (Darbyshire et al. , 1966 and Rondhuis,
1970).
Hyperimmune sera may confer some protection in
experimentally-infected calves. Research suggests that cattle may be
protected against severe lower respiratory disease by administering
hyperimmune sera intratrachially. Poor protection resulted when
hyperimmune serum was administered parenterally (Hartwell et al.,
1966 and Rondhuis, 1975).
Vaccination trials with bovine adenoviruses have been limited
to types 3 and 4. The first vaccination trial was conducted in Hungary
13
with bovine adenovirus type 4, using both a live and inactivated
vaccine (Bartha, 1967). The inactivated vaccine produced no adverse
side effects, but calves inoculated with the live virus vaccine experienced pyrexia, and the virus was spread to uninoculated control
calves. Both vaccines elicited a humoral response to bovine adeno-
virus type 4 and a low level response to type 5, but no response to
types 1, 2 and 3 (Rondhuis, 1975).
Oil adjuvant vaccines appear to be more effective in both the
initial antibody response and the duration of a demonstrable antibody
titer. A passively-acquired serum-viral neutralization antibody titer
of >1g16, or a passive hemagglutination antibody titer of >1:320 may
interfere with the response to the vaccine, while a serum neutralization antibody titer of <1:8, or a passive hemagglutination antibody titer
of <1:20 have little effect upon the immune response (Rondhuis, 1975).
Live viral vaccines generally produce antibodies more rapidly
and at a higher level than inactivated vaccines. However there are
several disadvantages to the use of a live viral vaccine. One disadvantage is the possibility of the vaccine containing an unwanted
virus (latent virus). Another disadvantage is that certain adenoviruses are oncogenic when inoculated into laboratory animals. What
possible risk this poses to humans or cattle is unknown. A third
disadvantage is that modified-live adenoviruses may be shed to
susceptible animals and may regain virulence. Live viral products
14
are generally not recommended for use in pregnant animals (Bartha,
1967; Darbyshire, 1966; and Rondhuis, 1970 and 1973).
Classification of the Family Adenoviridae
In 1975, the International Committee of Taxonomy of Viruses
divided the family Adenoviridae into two genera--Mastadenovirus
(adenoviruses of mammals) and Aviadenovirus (adenoviruses of birds)
(Fenner, 1976). This classification scheme replaced the system
described by Wildy, in which these viruses were classified in the
genus Adenovirus (Wildy, 1971). Presently 33 types of human adeno-
viruses are accepted, while two candidate human adnoviruses types
34 and 35 have not yet been officially accepted (Hilleman et al., 1954;
Keller et al. , 1977; and Stalder et al., 1977). Additional isolates of
adenoviruses from lower animals are recognized. These include
eight accepted types and several untyped isolates of bovine adeno-
viruses, five types and two untyped isolates of ovine adenoviruses
(Bauer et al., 1975; Mc Adair et al.
,
1976; McFerran et al., 1971;
and Sharp et al., 1974), four types of porcine adenoviruses
(Derbyshire et al.
,
1966; Haig et al. , 1964; Kasaza, 1966; and
Western Hemisphere Committee on Animal Virus Characterization,
1975), one type of equine adenovirus (McChesney et al., 1977), two
types of canine adenoviruses (Ditchfield et al., 1962 and Kapsenberg,
1959), twelve types of avian adenoviruses (McFerran et al. , 1977) and
15
twelve types of simian adenoviruses (Fenner, 1974 and Western
Hemisphere Committee on Animal Virus Characterization, 1975).
Additional adenoviruses have been isolated but not officially accepted
from quail (Olson, 1950), turkeys (Ahmed, 1971; Pereira, H.G. ,
1975; and Scott et al. , 1972), geese (Csontos, 1967 and Kaleta, 1969),
guinea fowl (Pascucci et al. , 1970), pheasants (Cakala, 1966),
pigeons, budgerigars, bantams and mallards (McFerran et al., 1976),
opossums (Morales-Ayala et al. , 1964), frogs (Clark et al. , 1973)
and goats (Dardiri et al. , 1977 and Gibbs, 1977). Serologic evidence
of previous adenoviral infection has also been found in domesticated
(Ahmed et al.
,
1968) and wild ducks (Masson et al.
,
1973).
Characteristics of Adenoviruses
Adenoviruses of all species have similar physical, chemical
and biologic characteristics. Like all cubic viruses, adenoviruses
have an icosahedral symmetry pattern. These viruses lack envelopes
and contain no essential lipid (Feldman et al. , 1961; Horsfall et al. ,
1965; Huebner et al., 1954; and Wildy, 1971). The particle size of
virions of most species ranges from 70-80 nm in diameter (excluding
projections). The virion contains 252 hollow cylindrical capsomeres:
240 nonvertex capsomeres (hexons), and 12 vertex capsomers (penton
base) with an extending fiber and knob (Horne et al. , 1963 and Wilner,
1969).
Electron microscopic studies show the hexons to be hollow
16
prolate ellipsoids with an inner diameter of 2.5 nm, and outer
diameter of 8-9.5 nm and a length of 8.8-11.5 nm (Pettersson et al.,
1967).
The penton base subunits have a globular structure with a
pentagonal outline when seen end-on (Pettersson et al., 1969). Fibers
of different adenovirus types vary in length considerably over a range
of 10-31 nm. Adenovirus types of the same subgroup have fibers of
equal length. All fibers have a terminal knob approximately 4 nm in
diameter (Norrby, 1969). The only exception is the avian adenovirus
CELO which has a characteristic double fiber structure with a knob
on each fiber (Laver et al., 1971).
All mammalian adenoviruses contain a single molecule of linear
double-stranded deoxyribonucleic acid of molecular weight 20-25 mil-
lion daltons, representing 11.6-13.5% of the virion mass. The
guanine + cytosine (G + C) content varies from 48-60%. Among the
human adenoviruses, the more oncogenic types have a lower G + C
content (Pina et al., 1965); the reverse is true for simian adenoviruses (Ginsberg, 1958).
Adenoviruses are good antigens. Serologic techniques have
identified group-specific, type -specific and subgroup-specific antigenic determinants on the hexon polypeptide; and group-specific and
subgroup-specific determinants on the fiber (Norrby, 1969 and 1971).
All adenoviruses except avian types and subgroup II bovine adeno-
viruses share a common complement-fixing antigen (Rowe et al.,
17
1962).
Adenoviruses are acid stable (Hamparian et al., 1963), rela-
tively sensitive to heat (Wallis et al. , 1962), and divalent cations
stabilize adenoviruses against heat inactivation in the pH range 4-6,
but not at more acidic or alkaline levels (Yamamoto, 1967).
Most adenoviruses agglutinate red blood cells from some
species of animals. Rat or monkey erythrocytes are generally used
in hemagglutination studies (Norrby, 1971 and Rosen, 1960).
The replication of human adenovirus type 5 serves as a good
model of the dynamics of replication. The replication cycle extends
over a prolonged period. The eclipse period varies with the cell-
viral system, but is usually between 13-18 hours (Green et al., 1970).
Attachment can take place at 0-4°C, although subsequent replication
must occur at approximately 37°C. The elongated fibers probably act
as contact points between the virus and cell receptors (Phillipson
et al., 1968 and Schlesinger, 1969).
Upon attachment, the adenovirus enters the cell by either
engulfment and phagocytosis (Chardonnet et al. , 1970), or direct
penetration (Chardonnet, 1972). The virion core, with little extrane-
ous protein, then enters the nucleus (Chardonnet et al., 1972 and
Longberg-Holm et al., 1969). Structural proteins comprising the
hexon, penton base, fiber and core proteins, and the nonstructural
proteins (including the T and P antigens and five nonstructural cellspecific polypeptides) are translated (Russell, 1972).
18
Intact virions, excess viral DNA, and protein comprise the
characteristic Cowdry type A or B inclusions which are found in
infected cells (Martinez-Palomo et al. , 1967 and Simmons et al. ,
1976).
Assembly of the virion is confined to the nucleus. Intact virions
are gradually released from the infected cell. Arginine is required
for virion production (Bonifas, 1967).
Adenoviruses characteristically replicate in epithelial cells in
cell culture (Ginsberg, 1958). The infected epithelial cells become
retractile, clumped and rounded (Hilleman et al., 1954 and Simmons
et al., 1976) and detach from the glass (Pereira, H. G. , 1958 and
Valentine et al., 1965). The infected cells increase their glucose
uptake, and as the glycolytic byproducts accumulate, the medium
becomes increasingly acidic (Fisher et al., 1957). Consequently,
adenoviral-infected cell cultures are more acidic than uninoculated
controls (Inaba et al., 1968).
The cytopathic action of adenoviruses results from the production of a protein involved in the early reversible clumping of cells and
virus multiplication, which is responsible for irreversible nuclear
changes (Pereira, H.
1958).
Adenoviruses can induce tumor formation in newborn hamsters
and rats. Human adenoviruses types 1,
3, 5, 7, 8, 12, 14, 16, 18,
21, 24, 31 (Huebner et al., 1954 and Trentin et al.
,
1962 and 1968),
19
simian adenoviruses types SA 7, SV 20, SV 33, SV 34, SV 37, SV 38
(Hull et al.
,
1965), avian adenovirus CELO (Sarma et al., 1965),
infectious canine hepatitis virus (Sarma et al. , 1967) and bovine
adenoviruses types 3 and 8 (Darbyshire, 1966 and Rondhuis, 1973)
have been shown to induce tumors in experimental animals. Some
types are more oncogenic that other types. No etiologic relationship
between natural tumors and adenoviral infection has been demonstrated (Gilden et al. , 1970 and McAllister et al. , 1972).
20
III. MATERIALS AND METHODS
Media
Growth medium for primary bovine embryonic kidney (BEK)
cell cultures consisted of Earle's balanced salt solution (EBSS), supplemented with 10-12% heat inactivated bovine serum, 1. 5% lactalbumin hydrolysate (LAH) and 4.44 mg /ml sodium pyruvate. Three
hundred units of penicillin and 30011g of streptomycin sulfate per ml
were added to the medium. Growth medium for secondary BEK cul-
tures and primary bovine testicular cell cultures was similar to the
above medium except that it was supplemented with 10-12% heat
inactivated donor calf serum.
Donor calf serum refers to serum from calves which were
raised in isolation and were free of antibodies to bovine adenoviruses.
Bovine serum was obtained from a local abbatoir, and was not tested
for antibodies to bovine adenoviruses. All sera were heat inactivated
at 56°C for 60 minutes prior to use.
Maintenance medium was similar to growth medium except
that it was supplemented with 3% heat inactivated donor calf serum.
Maintenance medium used in cultures containing first passage viral
isolation samples also contained 1001-1.g/m1 neomycin sulfate.
Earle's balanced salt solution supplemented with 0. 5% LAH,
100 units of penicillin and 100 lag streptomycin sulfate per ml, was
21
used to wash cell cultures prior to inoculation.
Virus isolation diluent (VID) consisted of Dulbecco phosphate
buffered saline (PBS), 1. 0% peptone, 0. 25% gelatin and 500 µg /ml of
neomycin sulfate.
Versene-trypsin solution (V-T) consisted of calcium and
magnesium-free PBS, 0. 15% trypsin,
1
0. 4% ethylenediamine tetra-
acetic acid (EDTA) and 0. 5% phenol red.
Cell Cultures
Primary Tr ypsinization
Fetal bovine kidneys were obtained from 4-9 month old fetuses
at an abbatoir. The kidneys were aseptically removed and held at
4°C while being transported to the laboratory. The kidneys were
surface sterilized by placing them in boiling water for 7-10 seconds,
and cooled in sterile distilled water at 4°C. The capsule was
3
removed and the remaining cortical epithelial tissue cut into 2-4 mm
cubes. The minced tissue was mixed with 150 ml of growth medium
previously warmed to 37 °C, and stirred constantly for ten minutes
to remove connective tissue, blood and cell debris. The liquid was
discarded and replaced with approximately 150 ml V-T, and the tissue
1
Difco Laboratory, Detroit, Michigan.
22
processed by the trypsin digestion method. After trypsinization, the
mixture was strained through sterile gauze. The final cell suspension
was suspended in EBSS, which was supplemented with 5% donor calf
serum, stored at 4°C and resuspended as needed for five days posttrypsinization.
Testicles from calves 2-12 weeks of age were removed
aseptically. The testicles were surface sterilized by immersing them
in boiling water for 7-10 seconds, and cooled in sterile 40°C distilled
water. The tunica vasculosa and tunica albuginea were cut and
separated to expose the stroma of the testis. The collecting tubules
were separated and discarded, and the remaining tissue minced,
washed and trypsinized using the same procedure described for primary BEK cell trypsinization.
Primary Culture Techniques
Primary BEK cells were resuspended in growth medium to a
concentration of 1.0 x
106
cells /ml, and 25 ml of this suspension
transferred into 75 cm 2 plastic flasks. 2 The cells were then incubated at 37°C for five days, at which time the cells were retrypsinized,
or fed with EBSS supplemented with 5% heat inactivated bovine serum.
2
HA 30.
Corning Glass Company, Corning, New York, catalog number
23
The cultures which were fed were then trypsinized 2-4 days later.
Alternatively, 1 ml of the primary BEK cell suspension was
resuspended to a concentration of 1.0 x
106
cells /ml and transferred
into 16 x 125 mm screw-capped tubes and incubated at 37°C.
The primary testicular cell suspension was resuspended in
growth medium to a concentration of 1.0 x
106
cells /ml and trans-
ferred into 16 x 125 mm screw-capped tubes.
Secondary Trypsinization
2
After each BEK culture was confluent in the 75 cm plastic
flasks, the spent growth medium was decanted and the cell sheet
washed three times with V-T solution. The cells were dispersed with
V-T solution and resuspended at a concentration of 3.75 x
105
cells /ml.
One ml was dispersed into each well of a multiwell plastic plate,
3
16 x 125 mm screw-capped tube, while 2 ml was dispensed into
Leighton tubes containing coverslips.
Vaccination and Sampling
The calves used in this study were raised on a 580-Holstein-
Friesian cow dairy, located near Corvallis, Oregon. All calves
raised on the premise were females. An average of two to three
3
Linbro multiwell culture plate, Flow Laboratories Incorporated, Hamden, Connecticut, catalog number 76-033-05.
or
24
calves were added to the calf-raising facility daily. Calves were
raised in groups of one or two until they were approximately two weeks
of age, at which time they were placed in groups of 20-30 calves.
The calves in the larger groups were segregated in different areas of
the facility according to age. Management practice, nutrition and
housing conditions were considered to be good. Calves in this herd
have a past history of pneumonitis. Bovine adenovirus type 3 was
isolated from several calves with this disease in February, 1977.
Samples Taken
Samples for virus isolation were taken from 42 calves. The
calves were randomly divided into two groups of 21 calves each, as
they were born. One group comprised the vaccinated animals, while
the other 21 calves were non-vaccinated controls. The vaccinated
group was given 5 ml of the bovine adenovirus type 3 experimental
vaccine intramuscularly, when they were first examined, and a repeat
injection (5 ml intramuscularly) 10-14 days later.
Samples for viral isolation were taken at the time of initial
observation, and 2, 6 and 10 weeks of age (i. e. , 4 and 8 weeks after
second vaccination) from the animals vaccinated with the bovine
adenovirus type 3 vaccine, and at the time of first sampling, 6 and 10
weeks of age from those calves which did not receive bovine adeno-
virus type 3 vaccine. Samples for viral isolation included a
25
combination conjunctival/nasal swab and a throat swab. Conjunctival/
nasal samples were taken with sterile dacron-tipped swabs first
inserted deep into the nasal cavity, and with the same swab rubbing
the conjunctival mucosa vigorously. The tip of the swab was broken
into a 10 x
75
mm screw-capped tube which contained
The tube was stored at the ambient temperature
4
ml of VID.
(15-25°C)
for
approximately 90 minutes prior to arrival at the laboratory. Upon
arrival the tubes containing the samples were frozen at -70°C.
Throat swabs were taken by rubbing the tonsillar fossa with cottontipped wooden swabs which were 30 cm in length. The swabs were
treated as described above.
At the time of each sampling,
20
ml of blood were taken from
each animal being examined. The blood was allowed to clot overnight
at room temperature, and then it was placed at
The serum was separated and stored at
4°C
for
2-18
hours.
-20°C.
Conjunctival/nasal and throat samples were stored for
1-8
days
before processing. The swabs were aseptically removed and the
residual fluid expressed. The remaining fluid was centrifuged at
755 x g for 15 minutes at
22-24°C.
used to inoculate cell cultures.
The supernatant was decanted and
26
Inoculation in Cell Culture
Prior to inoculation the cultures were washed three times with
EBSS, the wash solution decanted, and 0.2 ml of each test sample
inoculated onto the cell monolayers (1. e. , both BEK and bovine
testicular cell cultures). The inoculum was adsorbed for 45-60 minutes at 37 °C. During the adsorption period the cultures were rocked
8-10 times per min. on an oscillating platform. After adsorption,
1.0 ml of maintenance medium was placed in each tube. The cultures
were examined once every 3-5 days, and blind passaged at 10-14 days
post inoculation. All samples were blind passaged a minimum of four
times in both BEK and bovine testicular cell cultures. Each sample
was held for a minimum of 55 days in cell cultures.
Viral Pool Preparation
When cytopathic effect (cpe) was observed in the cell cultures,
the affected culture material was freeze-thawed and 0.2 nAl used to
inoculate additional cultures. Cultures showing cpe for a second
time were freeze-thawed (alternating temperatures of -20°C and
25°C) and centrifuged at 1085 x g for 15 min. at 4°C. The super-
natant was decanted into 2 dram vials and frozen at -70°C.
27
Viral Identification
The following test procedures were used to determine the
identity of each virus isolated from this study: (a) cytopathic effect,
including inclusion body characteristics, (b) sensitivity to chloroform, (c) viral ultrastructure, and (d) serum-viral neutralization
(or inhibition) tests.
CpoEathic Effect and Inclusion Body Characteristics
The progression and characteristics of cellular alterations
induced by each viral isolate were examined. Cell cultures were
prepared in Leighton tubes containing coverslips. Each culture was
inoculated with O. 4 ml of a viral isolate, and the inoculum allowed to
adsorb for 45-60 minutes at 37°C. Following adsorption, 2 ml of
maintenance medium was added to each tube, and the cultures incu-
bated at 37°C. When cpe first appeared, coverslips from cultures
with cellular alterations were removed and stained with either
acridine orange, or hemalotoxylin and eosin.
Coverslip cultures stained in acridine orange were rinsed once
briefly in PBS and held in Carnoy's fixative overnight. After fixation
the coverslips were processed through 50 and 30% ethanol (in descend-
ing order), and rinsed three times in citrate buffer, pH 3.6-3.8. The
coverslips were stained in a 0. 01% acridine orange solution, rinsed
28
three times in citrate buffer, mounted in buffer and observed
immediately using a dark field fluorescent microscope.
Covers lip cultures for hemalotoxylin and eosin staining were
rinsed once in PBS and fixed overnight in Zenker's solution. After
fixation the coverslips were washed for 5 min. in tap water, placed
in Lugol's solution for 5 min. , washed 2 min. in tap water and placed
in a 5% sodium thiosulfate solution for 5 min. The coverslips were
stained by the Harrison hemalotoxylin and eosin method.
Chloroform Sensitivity
Sensitivity to chloroform was determined for each viral isolate.
Reagent-grade chloroform (0. 05 ml) was added to each 1.0 ml of
viral solution (Feldman et at. , 1961). The mixture was shaken for
10 minutes at 4°C and centrifuged immediately at 1085 x g for 15
minutes at 4°C.
After centrifugation the aqueous layer was decanted
into a 25 ml erlenmeyer flask. The flask was held for approximately
30 minutes and shaken intermittently to allow excess chloroform to
evaporate. Chloroform-treated and non-treated suspensions of the
viral isolates were titered concurrently.
Serum-Viral Neutralization Test
The serum-viral neutralization test was used to identify those
viral isolates believed to be strains of bovine adenovirus type 3. Each
29
viral isolate was titered and the median tissue culture infectious
dose (TCID/50) determined by the Reed and Muench formula. Equal
amounts of unknown virus were added to serially diluted reference
antiserum to bovine adenovirus type 3. The mixture was incubated at
37°C for one hour. After incubation, 0.2 ml of the serum-viral mix-
ture was inoculated into each of five wells in a multi-well plastic
plate, which contained monolayer BEK cell cultures. The serum-
viral mixture was adsorbed for 30 minutes, and 1 ml of maintenance
medium supplemented with 50 units /ml mycostatin was dispersed
into each well. The plates were incubated at 37°C, in an atmosphere
of 3-5% CO2 for 12 days. The cultures were examined post-
inoculation days 7, 10 and 12. A positive test was regarded as an
inhibition of infectivity; i. e.
,
100 TCID
50
of virus was neutralized by
64 antibody units.
Viral Ultrastructure
One drop of a viral pooled preparation (0. 05 ml) of each isolate
was dispensed into each well of a spherical-bottom plastic plate.
The titer of the viral suspensions ranged from 1.8 x
102
to 5.6 x
4
105.
A Formvar coated 200-300 mesh grid was added to each well, and the
viral suspension allowed to dry onto the grid. The grids were stained
4
Microtiter, Cooke Manufacturing Company, Alexandria,
Virginia.
30
with 0.1 ml of a 3% solution of sodium phosphotungstate. Each grid
was observed in a transmission electron microscope.
5
Inoculation into Embryonated Chicken Eggs
One-fourth ml of each pooled preparation, tentatively identified
as bovine papular stomatitis virus, was inoculated onto the chorioallantoic membrane of 10 day old embryonated chicken eggs. The
eggs were incubated at 37°C for 3 to 5 days, at which time the
chorioallantoic membrane was harvested and observed for the formation of lesions. Vaccinia virus (known to produce lesions on the
chorioallantoic membrane) was inoculated in a similar fashion.
Challenge
To eliminate the possibility that any sample or lot of cells contained a noncytopathogenic strain of bovine diarrhea virus, the
terminal passage of each cell lot was tested for the presence of viral
interference substances. Three days after inoculating the fourth blind
passage, one tube from each sample lot was inoculated with 0.1 ml
(100-1000 TCID/50/0.1 ml) of a cytopathic strain of bovine diarrhea
virus. Each tube culture was examined for the acceptance of cpe
5
Phillips EM 300, Mount Vernon, New York (United States
Headquarters).
31
(i. e. , replication of bovine diarrhea virus and consequently no
interferring substances) 5-7 days post-inoculation.
Viral Isolation Attempt from the Experimental Vaccine
One ml of the bovine adenovirus type 3 experimental vaccine was
diluted 1:4 in PBS, and centrifuged at 4°C for 15 minutes at 480 x g.
The supernatant was then dialyzed in EBSS at 4°C. EBSS was changed
after 2, 8 and 16 hours. After dialysis, the supernatant fluid was
serially diluted and inoculated into screw-capped BEK cell cultures
as described above. Four blind passages were made for a total of
55 days in cell culture.
Serum-Viral Neutralization Titer
The serum-viral neutralization test was used to determine the
serologic response to vaccination (or infection). A reference strain of
bovine adenovirus type 3 (strain 5C) (Mattson, 1973a) was titered and
diluted to contain 100-300 TCID/50 per 0.1 mi. Test serum from the
42 experimental animals was heat inactivated (56°C for 60 minutes)
and prepared in two-fold dilution steps. Equal amounts of serum and
viral mixtures were added together, reacted and inoculated as
described above. Serum-viral neutralization endpoints were calculated by the Reed and Muench formula.
32
IV. RESULTS
Clinical Observations
Among the vaccinated calves, two experienced pneumonia
(dyspnea, or dyspnea associated with rales and muffled lung sounds),
nine experienced conjunctivitis and two experienced keratoconjunc-
tivitis (Table I). Among the control calves eleven developed
pneumonia, three conjunctivitis and eleven keratoconjunctivitis
(Table II). All clinical diagnoses were made by a veterinarian.
In
both the vaccinated, and unvaccinated control calves, the highest
incidence of disease occurred between the fourth and eighth week of
life. Eight of the 21 control calves died of respiratory-related
disease. None of the vaccinated animals died during the 13 week
observation period.
Viral Isolation
Bovine adenovirus type 3 was isolated from five of the 42
calves under investigation. Four isolates were recovered from
samples taken when the calves were first vaccinated, one at the time
of the second vaccination, and one three weeks after the second
vaccination.
Another virus, believed to be bovine papular stomatitis virus,
was recovered from seven of the 42 calves. In only one case were
33
Table I. Clinical history of vaccinated calves during the 13-week
observation period.
Animal
Number
R 75
R 76
R 77
Vaccination
1st vac.
2nd vac.
1st vac.
2nd vac.
1st vac.
2nd vac.
Age
3
4
1st vac.
2nd vac.
days
weeks
6.5
II
8
9
it
10
II
13
II
3
days
weeks
2
II
4
II
5
H
6
u
7,5
II
9
II
10
13
15
Il
3
days
weeks
2
3
R 78
Clinical
Observations
11
It
11
5
II
6
8
11
13
II
2
2
3
4.5
6
7
8
11
13
IT
days
weeks
II
II
II
II
II
H
II
Severity
N
N
N
N
N
N
N
N
N
C
C
C
N
N
N
N
N
2+
1+
1+
N
N
N
P
P
p
N
N
N
N
N
N
N
N
N
1+
3+
2+
34
Table I. Continued.
Animal
Number
R 79
Vaccination
1st vac.
2nd vac.
Age
3
2
1st vac.
2nd vac.
days
weeks
3
H
4.5
H
6
7
8
11
13
R 80
Clinical
Observations
H
H
11
H
11
N
C
C
N
N
C
N
N
N
Severity
-
2+
2+
1+
days
1.5 weeks
3
3
4.5
5.5
6.5
9.5
11
R 81
1st vac.
2nd vac.
days
1.5 weeks
3
3
4.5
5.5
6.5
9.5
11
R 82
1st vac.
2nd vac.
5
2
1+
II
11
II
days
weeks
3.5
II
5
II
6
7
12
H
II
II
N
N
N
N
C
N
K
K
K
N
2+
-
4+
3+
1+
35
Table I. Continued.
Animal
Number
R 83
Vaccination
1st vac.
2nd vac.
Age
1
3
35
u
5
II
6
7
10
12
R 84
1st vac.
2nd vac.
1
2
1st vac.
2nd vac.
week
weeks
5
II
1
2
u
It
II
N
N
N
N
N
N
N
N
N
N
C
N
C
N
N
N
II
u
ft
u
/I
days
weeks
4
u
5
II
6
II
9
II
12.5
1+
week
weeks
5
2
N
N
N
N
N
C
N
u
3
Severity
u
3.5
10
12
1st vac.
2nd vac.
10
If
6
7
R 86
u
II
3.5
6
7
10
12
R 85
week
weeks
Clinical
Observations
11
3+
2+
N
C
C
N
N
N
2+
R
1+
1+
36
Table I. Continued.
Animal
Number
R 87
Vaccination
1st vac.
2nd vac.
Age
1
1.5 weeks
1st vac.
2nd vac.
Severity
N
C
2+
3
II
N
4
II
K
u
N
N
N
N
5
R 88
day
Clinical
Observations
8
II
9.5
u
12.5
II
days
1.5 weeks
2.5 II
3.5 II
6.5 u
8.5 If
3
11.5
II
N
N
N
N
C
N
P
RC
R 89
1st vac.
2nd vac.
3
1.5 weeks
2.5 II
3.5 u
6.5 u
8.5 II
12
R 90
R 91
1st vac.
2nd vac.
1st vac.
2nd vac.
days
II
day
1.5 weeks
2.5 "
1
6
8
u
10
u
1
II
day
1.5 weeks
2.5 II
6
8
II
10
II
II
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
2+
1+
1+
37
Table I. Continued.
Animal
Number
R 92
Vaccination
1st vac.
2nd vac.
Age
1
day
1.5 weeks
2.5
6
8
11
R 93
1st vac.
2nd vac.
11
II
ri
day
1.5 weeks
6
8
11
1st vac.
2nd vac.
ft
1
2.5
R 94
Clinical
Observations
1
II
u
Il
II
day
1.5 weeks
2.5
11
6
8
11
R 95
1st vac.
2nd vac.
1
II
u
11
day
1.5 weeks
2.5 u
6
8
11
u
II
ft
Severity
N
N
N
N
N
N
N
N
N
N
C
2+
RC
N
N
N
N
N
RC
N
N
N
N
N
N
N = Normal
RC = Ruptured cornea or blind
C = Conjunctivitis
P = Pneumonia (Dypsnea, rales,
K = Keratoconjunctivitis
muffled lung sounds)
R = Rapid respiration (abnormally
high respiration rate upon exertion)
Severity of disease signs based on a scale of 1+ to 4+ with 1+
representing slight disease and 4+ respresenting severe disease signs.
38
Table II. Clinical history of control calves during the
13-week observation period.
Animal
Number
W 122
Age
3
2
W 123
II
6
II
7.5
II
8. 5
II
9.5
II
12
H
14
II
3
days
weeks
4
H
6
II
7. 5
8. 5
H
9.5
If
il
N
N
N
N
K
K, P
K, P
N
4
H
N
6
ff
7. 5
II
8.5
II
9. 5
H
P
P
P
P
P
11
If
Severity
N
N
N
N
N
N
N
N
N
days
weeks
3
2
W 125
days
weeks
4
2
W 124
Clinical
Observations
K, ND, D
4+
4+
2+
2+-3+
4+
4+
4+
4+
4+
days
weeks
N
N
3. 5
"
5
11
N
N
6
7
11
P
3+
H
P
3+
2
2
10
39
Table II. Continued.
Animal
Number
W 126
Age
3
2
3.5
5
6
7
10
12
W 127
Clinical
Observations
3
days
weeks
u
u
II
u
u
II
days
2.5 weeks
W 128
4
u
5
ft
6
9
11
u
II
days
1.5 weeks
2.5 II
3
4
u
5
u
6
9
11
W 129
u
3
5
W 130
-
3+
±
N
N
N
N
N
K, P
K, P
3+-4+
If
days
u
u
days
weeks
6
7
10
11
N
N
N
N
N
K
K
4+
5
5
±
N
N
R
6
3.5
4+
u
If
2
K
K
-
H
1.5 weeks
2.5 II
4
N
N
N
N
Severity
II
u
II
N
N
N
N
C
C
-
2+
1+
N
N
N
N
N
II
P
II
N
N
u
4+
3+
40
Table II. Continued.
Animal
Number
W 131
W 132
Clinical
Observations
Age
week
2. 5 weeks
1
3.5
II
5
11
N
N
N
N
Severity
-
6
II
C
1+
7
II
II
N
N
C
-
10
12
1
2
II
2+
week
weeks
3. 5
2+
5
3+
6
7
9
10
12
W 133
days
1. 5 weeks
3
II
K
4+
4
II
K, P
2+-3+
5
II
N
-
8
II
K, M
2+-3+
10
13
I,
K, P
Dead
3+
N
N
K
-
3
W 134
2+
II
days
1. 5 weeks
3
3
II
4
II
5
8
It
II
P
P
K, P
K, P
10
11
13
II
K, M
13+
if
Dead
-
3+
4+
4+
4+
3+
4+
41
Table II. Continued.
Animal
Number
W 135
Clinical
Observations
Age
Severity
days
1. 5 weeks
0
3
4
W 136
days
1. 5 weeks
0
3
W 137
ff
u
Dead
N
N
-
K, R
K, P
3+
4+
4+
4
II
5
If
K, P
8
10
u
u
N
N
13
u
K, M
13+
Dead
days
1. 5 weeks
0
2. 5
3. 5
6. 5
u
8.5
u
u
If
N
N
N
N
K
P
4+
4+
3+
11.5
W 138
days
1. 5 weeks
0
2. 5
3. 5
6. 5
If
M
If
N
if
8.5
P, R
P
Dead
3+
If
P, R, K
Dead
3+-4+
12
W 139
N
N
0
1
4
6
9
u
2+
4+
days
week
weeks
If
42
Table IL Continued.
Clinical
Observations
Animal
Numbe r
W 140
Age
0
1
4
Severity
days
week
weeks
6
9
11
W 141
0
1
4
6
9
11
W 142
0
1
4
days
week
weeks
11
Dead
days
week
weeks
2+
6
9
11
W 143
0
1
4
days
week
weeks
6
9
11
N = Normal
K = Keratoconjunctivitis
C = Conjunctivitis
P = Pneumonia (dypsnea, rales,
muffled lung sounds)
R :z Rapid respiration (abnormally
Dead
ND = Nasal discharge
D = Diarrhea
M = Malaise
high respiration rate upon
exertion)
Severity of disease signs based on a scale of 1+ to 4+, with
1+ representing slight disease and 4+ representing severe
disease signs.
43
both bovine adenovirus type 3 and bovine papular stomatitis virus
recovered from the same calf.
Bovine adenovirus type 3 was not isolated from the vaccine after
three blind passages in BEK cell cultures, for a total of 42 days in
cell culture.
Table III. Bovine adenovirus type 3 and bovine papular
stomatitis virus isolation results.
Animal
Number
Virus
Isolate
R 82
BAV 3
BAV 3
R 82
R 85
R 87
R 88
R 90
R 91
R91
BPS
BPS
BPS
BAV 3
BPS
BAV 3
BAV 3
BAV 3
BAV 3
BPS
BPS
Sample
Type
T
C/N
C/N
C/N
C/N
C/N
C/N
Isolation Time
Calf Age
Sample No.
3
5.
3
5
5
6
6
7
3
6
2
1.5
3
6. 5
T
1
C/N
1
T
1
weeks
1-5 days
1-5
1-5
1-5
C/N
1
6
weeks
C/N
C/N
6
BPS
C/N
6
C/N = Combination conjunctival and nasal sample.
T
= Throat sample.
BAV 3 = Bovine adenovirus type 3.
BPS
= Bovine papular stomatitis
R 93
W 125
W 129
W 142
Sample number 1 was taken at the time of the first vaccination; sample number 2 at the time of the second
vaccination; sample number 3, 3 weeks after vaccination;
sample number 5, 4 weeks after second vaccination;
sample number 6, 5 weeks after second vaccination.
44
Viral Characterizations Bovine Adenoviruses
Behavior in Cell Cultures
The first cytopathic effect resulting from adenoviral infection
appeared four or five days post-inoculation. Cellular alterations con-
sisted of rounding, enlargement and increased opacity of epithelial
cells (Figure 1). The cells remained attached to the glass. By ten
days post-inoculation the effected cells aggregated into irregular
masses and started to detach from the glass. Fibroblastic cells
appeared to be unaffected. Uninoculated cell culture controls
remained free of cellular alterations (Figure 2).
Early changes in hematoxylin and eosin stained preparations
consisted of an accumulation of fine eosinophilic granules in the
nucleus, which later grouped into an eosinophilic mass. Basophilic
areas developed within the mass, until the entire mass became more
basophilic. The mature inclusion was a large metachromatic mass,
surrounded by a halo (Figure 3). Uninoculated cell cultures were
free of accumulated nuclear masses.
Bright yellow-green fluorescence was seen in the area of the
mature inclusion when infected cells were stained with acridine
orange and observed with a microscope with darkfield fluorescence
illumination (Figures 4 and 5) .
45
Viral Titration and Serum-Viral Neutralization Tests
Titers of adenoviral pools which were prepared within three
passages of the original isolations ranged from 1.8 x
to 1.4 x
103 TCID
50
/1m1
104 TCID 50/1 ml.
Each bovine adenovirus isolate was neutralized by hyperimmune
rabbit serum to bovine adenovirus type 3. Sixty-four antibody units
were the highest dilution tested.
Electron Microscopy
Virions ranged in size from 70. 6 nm to 80.2 nm when seen in
negatively stained preparations of viral pool material of each of the
viruses isolated. One viral isolate R 87 C/N was determined to have
an icosahedral shape, 6 capsomeres on a triangular facet and a
capsomere number of 252 (Figures 6 and 7).
Viral Characterization: Bovine Papular
Stomatitis Virus
Behavior in Cell Cultures
The first cytopathic effect appeared two days post-inoculation
in bovine testicular cells. Cellular alterations consisted of clumping
and piling of cells. Cytopathic effect appeared to spread slowly from
localized foci of infection. Cells began to detach from the glass
46
causing spaces to develop 5-8 days post-inoculation (Figures 8 and 9).
Uninoculated testicular cell cultures remained free of cellular alterations (Figure 10).
In hematoxylin and eosin stained preparations the cytoplasm of
effected cells appeared granular. Many cells contained reddishpurple intracytoplasmic inclusions of various sizes and shapes.
In
some cells the cytoplasmic inclusion elongated, and surrounded the
nucleus in a horse-shoe configuration. Uninoculated cell culture con-
trols remained free of cellular alterations.
Yellowish-green fluorescence was seen in the area of the
intracytoplasmic inclusions, when viral-infected cells were stained
with acridine orange and observed using a dark-field fluorescence
microscope (Figures 11 and 12). Uninoculated testicular cell culture
controls did not contain fluorescent intracytoplasmic inclusions
(Figure 13).
Chloroform Sensitivity
All seven isolates believed to be bovine papular stomatitis virus
lost their infectivity when treated with chloroform. Virus controls,
which were not treated with chloroform, ranged in titer from
5.6 x
103
TCID 50/1 ml to 1.4 x
106 TCID 50/1 ml.
47
Replication in Cells of the Chorioallantoic Membrane
After three blind passages in embryonated chicken eggs, those
viruses believed to be bovine papular stomatitis virus did not produce
lesions on the chorioallantoic membrane. Control eggs which were
inoculated with vaccinia virus had characteristic lesions on the
chorioallantoic membrane.
Electron Microscopy
Brick-shaped virions that appeared to have extending surface
components were seen in negatively stained preparations (Figure 14).
Unlike vaccinia virus controls viewed simultaneously, the papular
stomatitis virions were approximately 255 to 262 nm in length, to
155 to 160 nm in width, while the vaccinia virions were 290 to 300 nm
in length and 230 to 240 nm in width.
Serologic Response to Vaccination and/or
Natural Infection
Fourteen of the 21 vaccinated calves experienced a four-fold or
greater rise in antibody titer to a reference strain of bovine adenovirus type 3. Of these 14 calves, two suffered from pneumonia, two
from conjunctivitis, two from keratoconjunctivitis and eight remained
clinically normal throughout the sampling period. Seven of the vac-
cinated calves produced less than a four-fold rise in antibody titer to
48
the reference strain of bovine adenovirus type 3, or a decline in titer.
Five of the seven calves had initially high antibody titers, while two
calves experienced less than a four-fold rise in humoral antibody titer
to the reference viral strain (Table IV).
No adverse side effects were
noted at the vaccination site in any of the vaccinated animals.
Seven of the 21 unvaccinated, control calves experienced a
four-fold or greater increase in humoral antibody titer to the reference strain of bovine andovirus type 3. Of these seven calves, four
suffered from pneumonia, one from keratoconjunctivitis, and two
remained clinically normal throughout the observation period. Three
of the seven calves died. Fourteen of the 21 unvaccinated control
animals produced either less than a four-fold rise in titer, a decline
in initial titer, or no change in humoral antibody titer. Of these 14
calves, three produced less than a four-fold response and four
experienced no change in titer to the bovine adenovirus type 3 refer-
ence strain.
49
Table IV. Viral-serum neutralization titer against bovine
adenovirus type 3, strain 5C: vaccinated calves.
Animal
Number
R 75
R 76
R 77
R 78
R 79
R80
R 81
R 82
R83
R 84
R 85
R 86
R 87
R88
R 89
R 90
R91
R 92
R 93
R 94
R 95
Sample Number
1
7.1
41
<1.4
<1.4
22
<1.4
2.8
<1.4
28
<1.4
<1.4
2
3
4
22
35
18
41
20
35
4.5
10
7
5.1
5.6
4.5
89
35
56
45
10
NS
<_ 1.4
4.5
<1.4
<1.4
NS
NS
22
5.1
22
45
5.6
5.6
5.6
5.6
2.8
7
1
56
41
45
NS
11
11
<1.4
2.8
<1.4
<1.4
<1.4
2.8
45
45
22
14
<1.4
2.8
<1.4
4.5
11
22
11
11
8.9
1
25
<1.4
<1.4
<1.4
<1.4
<1.4
_
2.8
11
28
NS
5.6
11
56
56
11
11
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS = Not sampled.
Sample number 1 was taken at the time of the first vaccination.
Sample number 2 at the time of the second vaccination.
Sample number 3 at 3-4 weeks after the second vaccination.
Sample number 4 at 6-8 weeks after the second vaccination.
Sample number 5 at 10-11 weeks after the second vaccination.
Titers expressed as the reciprocal of the highest serum
dilution which would neutralize 100 TCID50 virus.
50
Table V. Viral-serum neutralization titer against bovine
adenovirus type 3, strain 5C; control calves.
Animal
Number
W 122
W 123
W 124
W 125
W 126
W 127
W 128
W 129
W 130
W 131
W 132
W 133
W 134
W 135
W 136
W 137
W 138
W 139
W 140
W 141
W 142
W 143
= Calf dead.
Sample number 1
Sample number 2
Sample number 3
Sample number 4
Sample number 5
Sample Number
1
<1.4
5. 6
2
3
5. 6
2. 8
NS
2.8
<1. 4
<1. 4
<1. 4
<1.4
<1.4
5. 6
NS
<1.4
<1. 4
22
NS
NS
<1.4
<1.4
<1. 4
1. 6
5. 6
2. 8
2. 8
<1. 4
NS
11
1.6
2. 8
11
2. 8
5. 6
5. 6
2. 8
<1.4
<1.4
1. 6
2.8
<1. 4
<1. 4
5. 6
2. 8
NS
5. 6
2. 8
2. 8
<1. 4
2. 8
2. 8
NS
NS
NS
NS
NS
5. 6
NS
NS
NS
NS
NS
NS
NS
NS
4
5.6
2.8
<1.4
1.6
<1. 4
4. 5
<1. 4
<1. 4
<1.4
<1. 4
2. 8
<1.4
2. 8
5. 6
5
<1.4
<_1.4
5.6
NS
NS
NS
NS
<1. 4
NS
NS
NS
NS
5. 6
<1. 4
<1.4
NS
22
NS
NS
NS
2. 4
2. 8
2. 8
11
was taken at 0-1 week of age.
at 5-6 weeks of age.
at 7-8 weeks of age.
at 9-10 weeks of age.
at 11-13 weeks of age.
2. 8
1.4
NS
NS
51
Figure 1. Photomicrograph of cytopathic effect observed in bovine
embryonic kidney cell cultures 4-5 days post inoculation
with bovine adenovirus type 3 isolate R 87 C/N.
Magnification 86x.
Figure Z. Photomicrograph of a normal bovine embryonic kidney
cell culture. Magnification 86x.
52
53
Figure 3. Photomicrograph of a mature intranuclear inclusion body
observed in hematoxylin and eosin stained bovine
embryonic kidney cell culture 5 days after inoculation with
bovine adenovirus type 3 isolate R 87 C /N.
Magnification 400x.
Figure 4. Photomicrograph of a mature intranuclear inclusion body
observed in bovine embryonic kidney cell culture stained
with acridine orange 5 days after inoculation with bovine
adenovirus type 3 isolate R 87 C/N. Magnification 400x.
54
1
1
55
Figure 5. Photomicrograph of a normal bovine embryonic kidney
cell culture stained with acridine orange.
Magnification 400x.
Figure 6. Electron micrograph of three negatively stained bovine
adenovirus type 3 virions. Magnification 205,300.
56
I
57
Figure 7. Electron micrograph of a large group of negatively
stained bovine adenovirus type 3 virions.
Magnification 116, 000x.
Figure 8. Photomicrograph of early cytopathic effect observed in
bovine testicular cell cultures 2 ®3 days after inoculation
with bovine papular stomatitis virus isolate R 88 C /N.
Magnification 86x.
'
.../.
..
....
5
t
"
.
'
.
.
.4.
..!
4*'"
,
'`
°'
Of
:i
'1%.
4 ..
.
.. -.:
t°
'
.
.. .. q
7
'
...::":
;,
.:
11,
°
41
e.
....\.' .....)k;.:.,
.. .
xo,
. 4.,.....,
..,....71";k:,1;,.
.F..; i °I.'," .....,
..... ) ;
;1° '
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1' 7 ::?,
t
; . 11.". I16 ..' '... '''.- .":1/ .I
est.
.
. .... I* ....4.a.. L
,,,,e -
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.
44
:
4,
'14,1,17
''
-(.1
(7..1
"(.44'
'.
;
i, .. (:,.., a o',..;
Co.
0 .ifil,/,
.. ,
' ..
. 1. .....
k..
It ,,
I. '
I:,,,., V., i 01.'
,,-;
N, ,' Inv i.---'
..,.
.. ..
'
,
.
.
.
...,..
.
.,
a'
59
Figure 9. Photomicrograph of later cytopathic effect observed in
bovine testicular cell cultures 8 days after inoculation
with bovine papular stomatitis virus isolate R 88 C/N.
Magnification 86x.
Figure 10. Photomicrograph of a normal bovine testicular cell
culture. Magnification 86x.
60
61
Figure 11. Photomicrograph of a horse-shoe shaped intracytoplasmic
inclusion body observed in an acridine orange stained
bovine testicular cell culture 7 days after inoculation with
bovine papular stomatitis virus isolate R 85 C/N.
Magnification 155x.
Figure 12. Photomicrograph of a large intracytoplasmic inclusion
body observed in acridine orange stained bovine testicular
cell culture 7 days after inoculation with bovine papular
stomatitis virus isolate R 85 C/N. Magnification 155x.
N
,o
63
Figure 13. Photomicrograph of a normal acridine orange stained
bovine testicular cell culture. Magnification 155x.
Figure 14. Electron micrograph of two negatively stained bovine
papular stomatitis virions. Magnification 1499 600x.
64
65
V.
DISCUSSION
Clinical Observations
Between October 1977 and February 1978, a study was
undertaken to determine the role of bovine adenovirus type 3 in the
etiology of enzootic calf pneumonia, associated with conjunctivitis and
keratoconjunctivities. Initial disease signs included excessive
lacrimation, pyrexia, nasal discharge and cough.
As the disease
progressed affected animals exhibited malaise, low weight gain and
pneumonitis (dypsnea, rates and muffled lung sounds as determined
by stethoscopic examination).
Bovine adenovirus type 3 had been isolated from calves on this
premise the previous year. The continued expression of disease
offered a good opportunity to study the efficacy of an experimental
vaccine against bovine adenovirus type 3.
In the current study, bovine adenovirus type 3 was isolated
from five of the 42 (11.9%) calves being studied. This is only the
third reported isolation of bovine adenovirus type 3 in the United
States (Mattson, 1973). While this may seem to be a low isolation
percentage, previous reports describe similar results. Wilcox
reported 17 bovine adenoviral isolates from a combined total of 297
(5.7%) corneal, conjunctival and nasal samples (Wilcox, 1969). Of
these 17 isolates only one was from a conjunctival or nasal sample.
66
In a later study, Wilcox isolated a bovine adenovirus from 22% of the
animals examined (Wilcox, 1970). Aldasy isolated viruses from 17 of
92 nasal samples, and 4 of 7 conjunctival swabs (Aldasy et al. , 1965).
Cole isolated a bovine adenovirus from 7. 1% of the samples taken
(Cole, 1970). Only Mattson has reported a higher isolation rate
(Mattson, 1973).
Viral isolation results alone are not believed to represent a
true index of the incidence of infection with bovine adenoviruses.
Serologic studies suggest that the infection rate is high with all
serotypes studied (Mohanty, 1971).
In one two-year study
involving over 200 cattle entering an Oregon feedlot, the serologic
incidence of infection approached 85% (Mattson, personal communication).
Of the seven adenoviral isolates, four were from one to five-day
old calves. The recovery of an adenovirus from calves this young
was unexpected. Previous bovine adenoviral isolates were from
calves six, seven and ten days of age (Mattson, 1973). Aldasy et al. ,
made a similar observation in calves infected with bovine adenovirus
type 4. Clinical signs of infection were observed in calves two weeks
to four months of age. Younger calves displayed no outward symptoms
of infection (Aldasy et al., 1965).
Several possibilities exist insofar as the isolation of an
adenovirus from this young a calf: (1) Each calf was allowed to
67
suckle the dam in the first hours of life. During this period of contact
the adenovirus might have been transmitted from the dam to her calf,
before the calf was moved into a rearing pen. (2) Bovine adenoviruses
are extremely resistant to commonly used disinfectants. Although the
bedding was cleaned often, the virus could have been present on the
pen structure or feeding equipment, and transmitted to the calf
directly from this source. (3) Infection might have occurred in utero.
(4) Virus might have been spread from the older calves to the newborn
calves by aerosol. (5) Virus may have been spread by animal care-
takers.
Of these possibilities,
1,
2, 4 and 5 seem most probable. It
should be noted that the calf rearing area was not designed to hold the
number of calves present in the facility. Furthermore, bovine adenoviruses have been shown to be extremely resistant to dessication and
disinfectant action. Disinfection of feeding utensils and caretakers'
hands was not practiced in the dairy in question.
The possibility that the calves were infected in utero seems
more remote. Calves were always free from disease signs before
they were included in this study (even though they may have been in
the incubation stage of the disease). There have been some reports
however of bovine adenoviruses of the subgroup II classification being
involved in fetal infection (Dierks et al. , 1976). This has not been
shown with type 3 bovine adenoviruses. Mattson has suggested
68
previously that infected dams may revert to a viral shedding state
during the paranatal period (Mattson, 1973). The viral shedding pattern may be important in determining the time of calfhood infection.
Those isolates which were tentatively identified as bovine
papular stomatitis virus were all obtained from conjunctival /nasal
samples taken from calves six to seven weeks of age. The isolates
were tentatively grouped within the subgroup parapoxvirus, type
stomatitis papulosa (bovine 1) (Anonymous, 1973) based upon the fol-
lowing criteria (1) Electron micrographs revealed an ovoid virion
of size 255-263 nm in length by 155-160 nm in width. (2) Inoculation
of the virus onto the chorioallantoic membrane of embryonated
chicken eggs did not result in the production of lesions. (3) Infectivity
was lost after treatment with chloroform. (4) Cellular alterations
consisted of the formation of intracytoplasmic inclusion bodies of
various sizes and shapes. Based upon the acridine orange staining
reaction these inclusions were believed to contain deoxyribonucleic
acid.
Response to Vaccination
The incidence and severity of respiratory disease in the
vaccinated animals was less than in the control calves. Fourteen of
the 21 calves vaccinated against bovine adenovirus type 3, experi-
enced a four-fold or greater rise in serum-neutralizing antibody titer
69
to bovine adenovirus type 3, as measured between either the first or
second vaccination and four weeks post-vaccination. Of these 14
calves, two suffered from a comparatively mild case of pneumonia,
two from conjunctivitis, two from keratoconjunctivitis and eight
remained clinically normal.
Results from the vaccinated calves differ markedly from the
response to natural infection in the unvaccinated control calves.
Serologic response (as measured by the viral serum-neutralizing
antibody titer) to natural infection was not as marked as the response
to vaccination.
Bovine papular stomatitis virus was isolated from 17% of the
calves in this study, although the incidence of infection may have
exceeded the isolation percentage. Bovine papular stomatitis virus
has not been isolated from the lower respiratory tract of calves, nor
has it been recognized as an etiologic agent of calf pneumonia.
Accordingly, the high incidence of pneumonia in the control calves was
believed to be due to initial infection with bovine adenovirus type 3, or
bovine adenovirus type 3 in combination with secondary bacterial and
mycoplasmal infection.
No attempt was made to determine the incidence of infection with
Moraxella bovis. This bacterium is a recognized etiologic agent of
keratoconjunctivitis (Pugh et al.
,
1966 and 1971), and may have been
responsible for many of the severe cases of the disease observed in
70
the current study.
Infection with bovine adenoviruses has also been associated with
conjunctivitis and keratoconjunctivitis (Wilcox, 1970), however
research is lacking in this field. Some cases of severe keratoconjunctivitis may be caused by bacterial-viral interaction.
While some question may exist as to the role of infection with
bovine adenovirus type 3 in keratoconjunctivitis, it must be concluded
that the experimental vaccine appeared to prevent or eliminate clinical
manifestations of lower respiratory tract disease, if administered
under the proper conditions.
71
BIBLIOGRAPHY
Aghakhan, S.M. : Avian adenoviruses. Vet. Bull. 44:531-532, 1974.
El-Sisi, M. A. , El -Agroudi, M. A. , et al. s A
serological study of Newcastle disease and avian adenovirus
CELO infection in ducks. J. Egypt. Vet. Med. Assoc. 280 117 -
Ahmed, A. A. S
123, 1968.
Ahmed, A. A.S. CELO virus infections in turkeys (short note).
Berlklin. Wschr. 84:211-213, 1971.
Pneumoenteritis in calves
caused by adenoviruses. Acta. Vet. Acad. Sci. Hung.
Aldasy, P., Csontos, L., Bartha, A.
15:167-175, 1965.
Alterman, K., Embil, J., Easterbrook, K. B. , et al.: Liver
necrosis, adenovirus type 2 and thymic dysplasia. Virchows
Arch. Path. Anat. 360:155-171, 1973.
Anonymous: The WHO/FAO programme on comparative virology.
Vet. Rec. 93:434-440, 1973.
Isolation of adenovirus strains from calves
with virus diarrhea. Acta. Vet. Acad. Sci. Hung. 14:239-245,
Bartha, A., Aldasy, P.
1964.
Two further serotypes of bovine adenoviruses (serotypes 4 and 5). Acta. Vet. Acad. Sci. Hung.
Bartha, A., Aldasy, P.
160107 -108, 1966.
Bartha, A. Immunization experiments on calves with type 4 bovine
adenovirus. Acta. Vet. Acad. Sci. Hung. 17:209-216, 1967.
Bartha, A. , Mathe, S., Aldasy, P. Proposal for subgrouping of
bovine adenoviruses. Acta. Vet. Acad. Sci. Hung. 19:319-321,
1969.
New serotype 8 of bovine adenovirus. Acta. Vet. Acad. Sci. Hung. 20:399-400, 1970.
Bartha, A., Mathe, S., Aldasy, P.
Isolation of a virus from sheep
and its classification as a serotype of ovine adenoviruses.
Zbl. Vet. Med. 22:656-665, 1975.
Bauer, K., Muller, H., Gurturk, S.
72
Beale, J. A. , Doane, F. , Ormsby, H. L. Studies on adenovirus
infections of the eye in Toronto. Am. J. Opthtal. 43:26-31,
1957.
Becroft, D. M. O. Bronchiolitis obliterans, bronchioectasis and
other sequale of adenovirus type 21 infection in young children.
J. Clin. Path. 24:72-82, 1971.
Bettinotti, C. M. , Straub, 0.C.: Experimental infection in cattle
with human adenovirus type 1. Zbl. Vet. Med. I. Abt. Orig.
199:427-431, 1966.
Betts, A. 0. , Jennings, A. R. , Lamont, P. H. , et al. : Inoculation of
pigs with adenoviruses of man. Nature 193 :45-46, 1962.
Bibrack, B. , McKercher, D. G.: Serologic evidence for adenovirus
infection in California cattle. Am. J. Vet. Res. 32:805-807,
1971.
Bonifas, V. H. Time, course and specificity of the argenine requirement for adenovirus synthesis. Arch. Ges. Virusforsch.
20:20-28, 1967.
Brandt, C. D. , Kimm, H. W. , Vargosko, A. J. , et al. : Infections in
18, 000 infants and children in a controlled study of respiratory
disease. I. Adenovirus pathogenicity in relation to serologic
type and illness syndrome. Am. J. Epidem. 90:484-500, 1969.
Bullock, G.: An association between adenoviruses isolated from
simian tonsils and episodes of illness in captive monkeys.
J. Hyg., Cambr. 63:383-387, 1965.
Burki, F., Schlenka, G., Pichler, L.: Bovine adenovirus infections
associated with enzootic pneumoenteritis in Austria. Proc. 7th
Cong. World Assoc. Buiatrics, 1972, London, pp 70-74, 1973.
Burki, F.: Experimental adenovirus vaccines in cattle. JAVMA
163:897-900, 1973.
Cakala, A.: Isolation of the CELO virus from pheasants. Medycyna
Wet. 22:261-264, 1966.
Chany, C. , Lepine, P., Lelong, M., et al.: Severe and fatal
pneumonia in infants and young children associated with adeno-
virus infection. Am. J. Hyg. 67:367-378, 1958.
73
Chardonnet, Y. , Dales, S. g Early events in the interaction of adenoviruses with He La cells. L Penetration of type 5 and intracellular release of the DNA genome. Virol. 40g462-477, 1970.
Charadonnet, Y., Dales, S. g Early events in the interactions of
adenoviruses with He La cells. III. Relationship between the
ATPase activity in nuclear envelopes and transfer of core
materials; a hypothesis. Virol. 48g342-359, 1972.
Clark, H. F. , Michalski, F. , Tweedell, K.S. , et al. g An adenovirus
FAV-1 isolated from the kidney of a frog Rana pipiens. Virol.
51g392-400, 1973.
Clegg, T. G., Phillip, J. I. H. , Halliday, G. J. 2 The use of killed
vaccines in the control of virus pneumonia in calves. Proc.
7th Cong. World Assoc. Buiatrics, 1972, London, pp 75-85,
1973.
Cole, A. M. x Experimental adenovirus pneumonia in calves.
Austral. Vet. J. 47306-310, 1971.
Collier, J. R. , Chow, T. L. , Benjamin, M. M. , et al. g The combined effect of infectious bovine rhinotracheitis virus and
Pasteurella hemolytica on cattle. Am. J. Vet. Res. 210195198, 1960.
Conor, J. D. g Evidence for an aetiologic role of adenoviral infection
in pertussis syndrome. New Eng. J. Med. 283g390-394, 1970.
Crandell, R. A. , Conroy, J. D. g The isolation and characterization
of a strain of bovine papular stomatitis virus. Proc. 17th Ann.
Meet. Am. Assoc. Vet. Lab. Diag. , pp 223-234, 1974.
Darbyshire, J. H. , Dawson, P.S. , Lamont, P. H. , et al. g A new
adenovirus serotype of bovine adenovirus. 3. Comp. Path.
752327 -330, 1965a.
Darbyshire, J. H. g Association of adenoviruses with bovine respiratory disease. Nature 208g307-308, 1965b.
Darbyshire, J. H. , Jennings, A. R. , Dawson, P. S. , et al. x The
pathogenesis and pathology of infection in calves with a strain
of bovine adenovirus type 3. Res. Vet. Sci. 7g81-93, 1966a.
74
Darbyshire, J. H. Oncogenicity of bovine adenovirus type 3 in
hamsters. Nature 211:102, 1966b.
Dardiri, A. H. , De Boer, C. J. Hamdy, F. M. : Response of American
goats and cattle to peste de petitis ruminants virus. Proc. 19th
Ann. Meet. Am. Assoc. Vet. Lab. Diag., 1976, pp 337-344,
1977.
Davidson, A. P. , Stone, D. A multivalent calf virus pneumonia
vaccine. Proc. 7th Cong. World Assoc. Buiatrics, 1972,
London, pp 90-97, 1973.
Davies, D. H. , Humphreys, J.: Characterization of two strains of
adenoviruses isolated from New Zealand sheep. Vet. Micro.
2:97-107, 1977.
Derbyshire, J.B. , Clarke, M.G., Jes set, D. M. o Observations on
the fecal excretion of adenoviruses and enteroviruses in conventional and "minimal disease°' pigs. Vet. Rec. 79:595-599,
1966.
Dierks, R. E. , Smith, M. H. , Gollehon, D. o Isolation and characterization of adenoviruses from aborted fetuses and calves with the
weak calf syndrome. Proc. 19th Ann. Meet. Am. Assoc. Vet.
Lab. Diag. , 1976, pp 395-404, 1977.
Ditchfield, J. MacPherson, L. W. , Zbitnew, A.: Association of a
canine adenovirus (Toronto A26/61) with an outbreak of
laryngotracheitis (°kennel coughs). Canad. Vet. J. 3:238-247,
,
1962.
Du Bose, R. T. , Grumbles, L. C. , Flowers, A. I. : The isolation of a
nonbacterial agent from quail with a respiratory disease.
Poul. Sci. 37654-658, 1958.
Enjalbert, L. , Larenz, M. B. , Lapchine, L. , et al. o Virologie de
liquide cephalorachidien. Ann. Inst. Pasteur 102:153-173.
1962.
Faulkner, R. , Van Rooyen, C. E. o Adenovirus types 3 and 5 isolated
from the cerebrospinal fluid of children. J. Canada Med.
Assoc. 87:1123-1125, 1962.
Feldman, H. A. , Wang, S.S.: Sensitivity of various viruses to
chloroform. Proc. Soc. Exp. Biol. Med. 106:736-738, 1961.
75
Fenner, F. C. McAuslan, B. R. , Mims, C. A. , et al. : The biology
of animal viruses. 2nd ed. Academic Press, New York,
pp 12, 15, 16.
The biology of animal viruses. 2nd ed. Academic
Press, New York, pp 79-85.
:
Fenner, F.C.: The classification and nomenclature of viruses.
Summary of results of meetings of the international committee
on the taxonomy of viruses in Madrid, September 1975.
J. Virol. 71:371-378, 1976.
Fisher, T. N. , Ginsberg, H.S.: Accumulation of organic acids in cell
cultures infected with type 4 adenovirus. Proc. Soc. Exp. Biol.
Med. 95:47-51, 1957.
Franklin, R. M. , Harrison, S. L. Petters son, U. , et al. Structural
studies on the adenovirus hexon. Cold Spring Harbor Symp.
Quant. Biol. 36:503-510, 1972.
Encephalitis associated with adenovirus type 7 occurring in a family outbreak.
J. Pediat. 68:142-144, 1966.
Gab rielson, M. O. , Clifford, J.
Gibbs, C.J.
Hsuing, G. D.
Adenoviruses of goats. Res. Vet. Sci. 23:331-335,
1977.
Gilden, R. V. , Kern, J. , Lee, Y. K. , et al. : Serologic survey of
human cancer patients for antibody to adenovirus T-antigen.
Am. J. Epidem. 91:500-509, 1970.
Gillespie, J. H.: Comments on bovine adenovirus infection. JAVMA
163:901, 1973.
Ginder, D. R.: Increased susceptibility of mice infected with mouse
adenovirus to Escherichia coli-induced pyleonephritis. J. Exp.
Med. 120:1117-1128, 1964.
Ginsberg, H.S. : Characterization of the adenoviruses. III. Reproductive cycle of types 1 to 4. 3. Exp. Med. 107:133-152, 1958.
Gooheart, C. R.: DNA density of oncogenic and non-oncogenic simian
adenoviruses. Virol. 44:645-648, 1971.
76
Green, M. , Parson, P. T. , Pina, M. , et al. : Transcription of
adenovirus genes in productively infected and in transformed
cells. Cold Spring Harbor Symp. Quant. Biol. 35:803-818,
1970.
Pereira, M.S. : Isolation of an adenovirus from a pig. J. Comp. Path. 74:81-84, 1964.
Haig, D. A. , Clarke, M. C.
,
, Hilleman, M. R. , Ketler, A.
Contributions to
characterization and classification of animal viruses. Proc.
Soc. Exp. Biol. Med. 112:1040-1050, 1963.
Hamparian, V. V.
Haralambier, H. Azev, G.: Isolierung von rinderadenovirus bei
enzootischer cuffing - pneumonia der kalber. Arch. Exp. Vet.
Med. 23:1035-1042, 1969.
,
Hartley, J. W. , Rowe, W. P. A new mouse virus apparently related
to the adenovirus group. Viral. 11:645-647, 1960.
Adenoviruses in
blood clots from cases of infectious hepatitis. Science 152:
Hartwell, W. V. , Love, G. J. , Eidenbock, M. P.
1390, 1966.
Siem, R. A.: Viruses isolated from children with
infectious hepatitis. Am. J. Epidem. 84:495-509, 1966.
Hatch, M. H.
,
Heck, F. C. Jr. , Sheldon, W. G.
Pathogenesis of
, Gleiser, C. A. :
experimentally produced mouse adenovirus infection in mice.
Am. J. Vet. Res. 33:841-846, 1972.
Hierholzer, J. C. , Nuzhet, 0. A. , Gwaltney, J. M.: New human
adenovirus isolated from a renal transplant recepient: Description and characterization of candidate adenovirus type 34. J.
Clin. Micro. 1:366-376, 1977.
Werner, J. R.: Recovery of a new agent from
patients with acute respiratory illness. Proc. Soc. Biol. Med.
Hilleman, M. R.
,
85:183-188, 1954.
Hillis, W. D. , Holmes, A. W. , Davidson, V.: Serologic characteriza-
tion of adenoviruses isolated from chimpanzees with viral
hepatitis. Proc. Soc. Exp. Biol. , N. Y.: 129:366-369, 1968.
77
Hillis, W. D. , Garner, A. C. , Hillis, A. I. A new simian adenovirus
serologically related to human adenovirus type 2 from a
chimpanzee with viral hepatitis. Am. J. Epidem. 90:344-353,
1969.
Horne, R. W. , Wildy, P. : Virus structure revealed by negative
staining. Adv. Virus Res. 10:101-170, 1963.
Horsfall, F. L., Jr. Tamm, I. (eds): Viral and rickettsial diseases
,
of man. 4th ed., J.B. Lippincott Company, Philadelphia,
p 864, 1965.
Huebner, R. J. Rowe, W. P. , Ward, T. G. , et al. : Adenoidalpharyngeal-conjunctival agents. A newly recognized group of
,
viruses of the respiratory system. New Eng. J. Med. 259:
1077-1086, 1954.
Hull, R. N. , Johnson, I. S.
Culbertson, C. G. , et al. : Oncogenicity
of the simian adenoviruses. Science 150:1044-1046, 1965.
,
Hull, R. N. : The simian viruses. Virol. Monogr. , vol. 2, pp 1-66
(Springer, Vienna 1968).
Ide, P. R. , Thompson, R. G. , Ditchfield, W. J. B.: Experimental
adenovirus infection in calves. Canada J. Comp. Med. 33:1-9,
1969.
Ide, P.R.: The etiology of enzootic pneumonia in calves. Canad.
Vet. J. 11:194-202, 1970.
Tanaka, Y., Sato, K. et al. : Bovine adenovirus. II.
A serotype Fukuroi, recovered from Japanese cattle. Japan.
J. Microbiol. 12219-229, 1968.
Inaba, Y.
,
Jen, K. F. , Tai, Y., Lin, Y. C. et al.: The role of adenoviruses in
the etiology of infantile pneumonia and pneumonia complicating
measles. Chinese Med. 3. 81:141-148, 1962.
,
Jensen, R.: Scope of the problem of bovine respiratory disease in
beef cattle. JAVMA 152:720-723, 1968.
Kaleta, E. F. a CELO virus in goslings. Case report. Dt. Tierarztl.
Wschr. 76:427-428, 1969.
78
Kapsenberg, J. G.: Relationship of infectious canine hapatitis virus
to human adenovirus. Proc. Soc. Exp. Biol. , N. Y. 101 :611 614, 1959.
Kasza, L. Isolation of an adenovirus from the brain of a pig. Am.
J. Vet. Res. 27:751-758, 1966.
Keller, E. W. , Rubin, R. H. , Black, P. H. , et al. : Isolation of
adenovirus type 34 from a renal transplant recipient with interstitial pneumonia. Transplantation 23:188-191, 1977.
Kjellen, L. , Sterner, G. , Svedmyr, A.: On the occurrence of
adenoviruses in Sweeden. Acta. Pediat. Scand. 46:164-176,
1957.
Klein, M. , Earley, E. , Zellat, J.: Isolation from cattle of a virus
related to human adenovirus. Proc. Soc. Exp. Biol. Med.
102:1-4, 1959.
Klein, M. , Zellat, J. , Michaelson, T. C.: A new bovine adenovirus
related to human adenoviruses. Proc. Soc. Exp. Biol. Med.
105:340-342, 1960.
Layer, W. G. , Younghusband, H. B. , Wrigley, N. G.: Purification
and properties of chick embryo lethal orphan virus (an avian
adenovirus). Virol. 45:598-614, 1971.
Lon.gberg-Holm, K. , Phillipson, L.: Early events of the virus-cell
interaction in an adenovirus system. J. Virol. 4:323-338, 1969.
Maizel, J. V. , White, D. O. , Scharff, M. D.: The polypeptides of
adenoviruses. II. Soluble proteins, cores, top components and
the structure of the virion. Virol. 36:126-136, 1968.
Martinez -Palomo, A., LeBuis, J., et al.: Electron microscopy of
adenovirus type 12 replication. I. Fine structural changes in
the nucleus of infected KB cells. 3. Virol. 1:817-829, 1967.
Mathur, B. B. L. , Verma, K. C. , Agarwal, K. K. , et al. : Serologic
survey for the detection of common respiratory infections of
migrating birds; a note. Indian J. Animal Sci. 42:144-145,
1972.
Mattson, D.E. : Naturally occurring infection in calves with a bovine
adenovirus. Am. J. Vet. Res. 34:623-629, 1973a.
79
Mattson, D.E.
Adenovirus infections in cattle. JAVMA 163:894-896,
1973b.
Mattson, D.E. : Personal communication, 1978.
Mattson, D. E. Smith, P. P. Schmitz, J. A. , et al. : Pathogenesis
of type 4 bovine adenovirus. 1. Virological studies of the
acute disease phase (in press).
,
Matumoto, M.
,
Inaba, Y. , Tanaka, Y. , et al.: New serotype 7 of
bovine adenovirus. Japan. J. Microbial. 14:430-431, 1970.
,
Mayr, A. Wizigmann, B., Bib rack, B.
A bovine adenovirus
, et al. :
isolated from the lymph nodes of cattle. Arch. Ges. Virusforsch. 29:271-273, 1970.
Mc Adair, B. McC. , Mc Ferran, J. B.: Comparative serologic studies
with mammalian adenoviruses. Arch. Virol. 51:319-325, 1976.
McAllister, R. M. Gilden, R. V. , Green, M. : Adenovirus es in
human cancer. Lancet i:831-833, 1972.
McChesney, A. E. , England, J. J. , Adcock, J. L. et al. : Adenoviral
infection in suckling Arabian foals. Path. Vet. 7:547-564, 1970.
,
McChesney, A. E. , England, J. J. , Rich, L. J.: Adenoviral infection
in foals. JAVMA 162:545-549, 1973.
McChesney, A. E. England, 3. J. , Baratt, R. M. , et al. , : Serologic
comparison of seventeen equine adenoviruses isolated in the
United States. Austral. Vet. J. 1:410-412, 1977.
,
McFerran, J. B. , Connor, T. J. , McCracken, R. M. Isolation of
adenoviruses and reoviruses from avian species other than
domestic fowl. Avian Dis. 20:519-524, 1976.
McFerran, J. B. , McAdair, B. McC.: Avian adenoviruses -A review.
Avian Path. 6:189-217, 1977.
McKercher, D. G.: Bovine respiratory infections. JAVMA 152:729737, 1968.
Medical Research Council: A collaborative study of the etiology of
acute respiratory infection in Britain, 1961-4. British Med.
J. 2:319-326, 1965.
80
Merigan, T. C.: Host defenses against viral disease. New Eng. Med.
J. 290:323-329, 1974.
Moffatt, R.E. Schiefer, B.: Adenovirus infection in swine. Proc.
17th Ann. Meet. Vet. Lab. Diag. , pp 93-98, 1974.
Mohanty, S. B. , Lillie, M.G.: Experimental infection of calves with
a bovine adenovirus. Proc. Soc. Exp. Biol. Med. 120 :679
682, 1965.
Mohanty, S. B. : Comparative study of bovine adenoviruses
Vet. Res. 32:1899-1905, 1971.
Am. J.
Morales -Ayala, F. Nunes -Montiel, 0. , Merino, F. , et al. : Spontaneous isolation and characteristics of a new possum adenovirus. Bact. Proc. 117:Abst. VII, 1964.
,
Morax, U.: Note sur un diplobaccile pathogene pour le conjunctivae
humaine. Ann. Inst. Pasteur 10:337-345, 1896.
Morgen, C. , Rosenkranz, H.S., Mednis, B.: Structure and development of viruses as observed in the electron microscope. X.
Entry and uncoating of adenovirus. J. Virol. 4:777-796, 1969.
Mufson, M. A. , Zoller, L. M.
Adenovirus
, Mankad, V. N. , et al. :
infection in acute hemorrhagic cystitis: a study in 25 children.
Am. J. Dis. Childr. 121:281-285, 1971.
Mufson, M. A.
Belishe, R. B. Horrigan, T. J. , et al. : Association
of adenovirus and E. coli with acute hemorrhagic cystitis (AHC)
in children. J. Clin. Investig. 52:58a, 1973.
,
,
Neva, F. A. , Enders, J.F. : Isolation of a cytopathogenic agent from
an infant with a certain disease resembling roseola infantum.
J. Immunol. 86:128-132, 1961.
Norrby, E.: The structural and functional diversity of adenovirus
capsid components. J. Gen. Virol. 5:221-236, 1969.
Norrby, E.: Adenoviruses. In Comparative Virology,
(K. Maramorosch and E. Kurstak, eds. ), Academic Press,
New York, p 105, 1971.
81
Olson, N. O. : A respiratory disease (bronchitis) of quail caused by a
virus. Proc. U.S. Livestock Sci. Assoc. 54th Ann. Meet. ,
pp 171-174, 1950.
Parsons, J. T., Green, M.: Biochemical studies on adenoviral
multiplication. XVIII. Resolution of early virus-specific RNA
species in Ad 2 infected and transformed cells. Virol. 45:154162, 1971.
Pascucci, S. Quaglio, G. L., Maestrini, N. , et al.: Pancreitis due
to CELO virus infection in guinea-fowl. Atti. Soc. Ital. Sci.
Vet. 24:667-668, 1970.
,
Pereira, H.G. Kelly, B.: Latent infection of rabbits by adenovirus
,
type 5. Nature 180:615-616, 1957.
Pereira, H. G. : A protein factor responsible for the early cytopathic
effect of adenoviruses. Virol. 6:601-611, 1958.
Pereira, H. G. Allison, A. C. , Niven, J. S. F.: Fatal infection of
newborn hamsters by an adenovirus of human origin. Nature
,
96:244-245, 1963.
Pereira, H. G. : Adenoviruses of man and animals. Dev. Biol. Std.
28:28-41, 1975.
Pereira, M. S. , Candeias, J. A. W.: The association of viruses with
clinical pertussis. J. Hyg. Cambr. 69:399-403, 1971.
Pereira, M.S.: Adenovirus infections. Postgrad. Med. J. 49:798801, 1973.
Pettersson, U., Phillipson, C., Hoglund, S. : Structural proteins
of adenoviruses. I. Purification and characterization of the
adenovirus type 2 hexon antigen. Virol. 33:575-590, 1967.
Petters son, U. , Hoglund, S.: Structural proteins of adenoviruses.
III. Purification and characterization of adenovirus type 2
penton antigen. Virol. 39:90-106, 1969.
Phillip, J.I. H. , Omar, A. R. , Popovici, V., et al.: Pathogenesis
and pathology in calves of infection by bedsonia alone and by
bedsonia and reovirus together. J. Comp. Path. 78:89-99,
1968.
82
Phillip, J. I. H. : Virus pneumonia in calves. Vet. Rec. 86:280-284,
1970.
Phillip, J. I. H. , Sands, J. H.: The isolation of bovine adenoviruses
serotypes 4 and 7 in Britain. Res. Vet. Sci. 14:386-387, 1972.
Phillip, J.I. H.: The development of bovine respiratory diseases
vaccines. Prog. Immunobiol. Std. 5:304-308, 1972.
Phillipson, L. Longberg-Holm, K. , Pettersson, U.: Virus-receptor
interaction in an adenovirus system. J. Virol. 2:1064-1075,
,
1968.
Pina, M. Green, M.: Biochemical studies on adenovirus multiplication. IX. Chemical and base composition analysis of 28
human adenoviruses. Proc. Nat'l. Acad. Sci. U.S. 54:547-551,
,
1965.
Pugh, G. W. Jr. , Hughes, D. E. , McDonald, T. J.: The isolation and
characterization of Moraxella bovis. Am. J. Vet. Res.
27:957-962, 1966.
Pugh, G. W. Jr. , Hughes, D. E. McDonald, T. J.: Bovine infections
keratoconjunctivitis: Serological aspects of Moraxella bovis
infection. Am. J. Vet. Res. 35:161-166, 1971.
Rondhuis, P.R.: A new bovine adenovirus. Arch. Ges. Virusforsch
25:235-236, 1968.
Rondhuis, P.R.: Bovine adenoviruses. A comparative serological
and experimental study. Drukkerij-Uitgeverij G. van Dijk N.V.,
Breukelen, Netherlands, 1970.
Rondhuis, P.R.: Induction of tumors in hamsters with a bovine
adenovirus strain (serotype 8). Arch. ges. Virusforsch
107:147-149, 1973.
Rondhuis, P.R.: Bovine adenoviruses. A review of vaccination
experiments. Dev. Biol. Std. 28:493-500, 1975.
Rosen, L.: A hemagglutination-inhibition technique for typing adeno-
viruses. Am. J. Hyg. 71:120-128, 1960.
83
Rowe, W. P. , Huebner, R. J. , Gilmore, L. K. , et al.: Isolation of a
cytopathogenic agent from human adenoids undergoing spontan-
eous degeneration in tissue culture. Proc. Soc. Exp. Biol.
Med. 84:570-573, 1953.
Rowe, W. P. , Huebner, R. J. , Hartley, J. W. , et al. : Studies on the
adenoidal-pharyngeal-conjunctival (APC) group of viruses.
Am. J. Hyg. 61:197-218, 1955.
Rowe, W. P. , Hartley, J. W. : A general review of the adenoviruses.
Ann. New York Acad. Sci. 101:466-474, 1962.
Rus sell, W. C. , Skehel, J. J.: The polypeptides of adenovirus-
infected cells. J. gen. Virol. 15:45-57, 1972.
Sarma, P.S. , Huebner, R. J. , Lane, W. T.: Induction of tumors in
hamsters with an avian adenovirus (CELO). Science 149:1108,
1965.
Sarma, P.S. , Vass, W., Huebner, R. J. , et al.: Induction of tumors
in hamsters with infectious hepatitis virus. Nature 215:293294, 1967.
Schlesinger, R. W.: Adenoviruses: The nature of the virion and of
controlling factors in productive or abortive infection and
tumorigenesis. Adv. Virus Res. 14:1-61, 1969.
Scott, M., McFerran, J. B.: Isolation of adenoviruses from turkeys.
Avian Dis. 16:413-420, 1972.
Sharp, J. M.
,
McFerran, J.B., Rae, A.: A new adenovirus from
sheep. Res. Vet. Sci. 17:268-269, 1974.
Shefki, M.D., Benge, W. P. J.: A trivalent vaccine against virus
pneumonia in calves. Vet. Rec. 85:582-583, 1969.
Sickira, V. P. Pile, E. R. , Dragarov, J. G.: Infectivity of bovine
adenoviruses for cattle. Abstract Vet. Bull. 42: no. 2472,
,
1972.
Simmons, D. G. , Miller, S. E. , Gray, J. G. , et al. : Isolation and
identification of a turkey respiratory adenovirus. Avian Dis.
20:65-74, 1976.
84
Scholes, S. : Current considerations
in public health of the role of animals in relation to human viral
disease. JAVMA 136:481-485, 1960.
Sinha, S. K. , Fleming, L. W.
Stalder, H. , Hierholzer, J. C. , Oxman, M. N.: New human adenovirus (candidate adenovirus type 35) causing fatal disseminated
infection in a renal transplant patient. J. Clin. Microbiol.
6:257-265, 1977.
Sussenbach, J.S. : Early events in the infection process of adenovirus type 5 in HeLa cells. Virol. 33:567-574, 1967.
Swann, N.H.: Acute thyroiditis. Five cases associated with adenovirus infection. Metabolism 13:908-910, 1964.
Tanaka, Y., Inaba, Y., Ito, Y., et al.: Bovine adenovirus. I.
Recovery of a serotype, Nagano, from Japanese cattle. Japan.
J. Microbiol. 12:77-95, 1968.
Thabaut, A. , Laverdaut, C., Bertain, J., et al.: Contribution a 1
etude des adenovirus. Adenovirus et syndrome de FeissingerLeroy-Reiter. Rev. dt Immunol. 26:335-343, 1962.
Teng, C.H. Adenovirus pneumonia epidemic among Peking infants
and pre-school children in 1958. Chinese Med. J. 80:331-339,
1960.
Trentin, J. J. , Yabe, Y., Taylor, G.: The quest for human cancer
virus. Science 137:825-841, 1962.
Trentin, J. J. , Van Hoosier, G. L. , Jr. , Samper, L.: The oncogenicity of human adenoviruses in hamsters. Proc. Soc. Exp.
Biol. Med. 127:683-689, 1968.
Valentine, R. C. , Pereira, H. G.: Antigens and substructure of the
adenoviruses. 3. Mot. Biol. 13:13-20, 1965.
Van der Veen, J.: Adenoviruses and viral pneumonia in children.
J. Hyg. Epidem. Microbiol. Immunol. 6:85-88, 1962.
Walker, W. A. , Is selbacker, K. J. , Block, K. J.: Intestinal uptake of
macromolecules: Effect of oral immunization. Science 177:
608-610, 1972.
85
Wallis, C., Yang, C., Melnick, J. L.: Effect of cations on thermal
activation of vaccinia, herpes simplex and adenoviruses. J.
Immunol. 89:41-46, 1962.
Western Hemisphere Committee on Animal Virus Characterization:
An updated listing of animal reference virus recommendations.
Am. J. Vet. Res. 36:861-872, 1975.
Wilcox, W. C. , Ginsberg, H.S., Anderson, T. F.: Structure of type 5
adenovirus. I. Antigenic relationship of virus-structural pro-
teins to virus-specific soluble antigens from infected cells.
J. Exp. Med. 118:295-306, 1963.
Isolation of adenoviruses from cattle with conjunctivitis and keratoconjunctivitis. Austral. Vet. J. 45:265-270,
Wilcox, G. C.
1969.
Wilcox, G. C.: The aetiology of infectious bovine keratoconjunctivitis
in Queensland. 2. Adenovirus. Austral. Vet. J. 46:415-420,
1970.
Wildy, P.: Classification and nomenclature of viruses: first report
of the International Committee on Nomenclature of Viruses.
Monogr. in Virol. 5:1, 1971.
Wilner, B.I.: A classification of the major groups of human and
other animal viruses. 4th ed. Burgess Publishing Co. ,
Minneapolis, 250 p., 1969.
Yamamoto, T. The effect of ion concentration and pH on the thermal
stability of a canine adenovirus. Can. J. Microbiol. 13:11391149, 1967.