Download Cell-mediated immune responses in cattle

Retrospective Theses and Dissertations
1975
Cell-mediated immune responses in cattle
Carlos Reggiardo
Iowa State University
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Reggiardo, Carlos, "Cell-mediated immune responses in cattle " (1975). Retrospective Theses and Dissertations. Paper 5503.
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Xerox University EiAicrofiSms
300 North Zeeb Road
Ann Arbor, Michigan 481OS
I
76-1868
REGGIARDO, D.V.M., Carlos, 1944CELL-MEDIATED IMMUNE RESPONSES IN CATTLE.
Iowa State University, Ph.D., 1975
Veterinary Science
Xerox University l\/licr0films,
Ann Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
Cell-mediated immune responses in cattle
by
Carlos Reggiardo
A Dissertation Submitted to the
Graduate Faculty in Partial Fulfillment of
The Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Department:
Major:
Veterinary Microbiology
and Preventive Medicine
Veterinary Microbiology
Approved:
Signature was redacted for privacy.
Signature was redacted for privacy.
'For the Major Department
Signature was redacted for privacy.
Iowa State University
Ames, Iowa
1975
11
TABLE OF CONTENTS
Page
INTRODUCTION
1
REVIEW OF LITERATURE
3
Resistance to Viral Infection
3
Immunosuppression During Viral Infections
40
Iimune Responses in Cattle to IBR and BVD Viruses
46
MATERIALS AND METHODS
48
General Methods
48
Experimental Procedures
53
RESULTS
62
BVD Virus Infection
62
IBR Virus Infection
77
DISCUSSION
89
Recovery from Infection
89
Effect of BVD Virus Infection on the Iimune System
98
SUMMARY
103
BIBLIOGRAPHY
105
ACKNOWLEDGMENTS
129
1
INTRODUCTION
Viruses play an extremely important role in the direct causation of
disease by interacting with host cells through an extreme variation of
pathogenic mechanisms ranging from cytolytic effects to integration of
viral genomes with the host's genetic material.
The virus-host cell
interaction can also result in other disease processes in which the virus
seems to play an indirect role.
The interaction of viruses with the
cells, tissues and humoral products of the imnune system constitute the
most important of these indirect effects, manifested through inhibition of
iimune responses or indirect tissue damage by antiviral immune mechanisms.
The immunosuppressive potential of viral infection has been recog­
nized for over half a century but only in the last 10 or 15 years has it
been the subject of intensive investigation.
A variety of viruses be­
longing to several groups and including both oncogenic and nononcogenic
viruses have been shown to be immunosuppressive.
Ability to infect and
alter the cells of the immune system is perhaps the common denominator of
most of these immunosuppressive viruses.
Bovine viral diarrhea (BVD) virus has been the subject of appreciable
investigation at Iowa State University and elsewhere.
The nature of the
disease produced and the pathogenesis of the infection have been investi­
gated, but the virus-host interrelationships concerned with immunologic
processes have not received concerted study.
effect upon lymphoid tissues.
The virus elicits a marked
Field observation of post-vaccinal reac­
tions (Peter et al., 1967) as well as studies on vir/us-lymphoid cell
interaction in vitro (Truicc and ShéciiïnèlstÊr, 1973; "liscoplatt st &1.,
2
1973a) have suggested the possibility of immunodepression during infec­
tion, but detailed studies on the BVD virus-host irrenune system interac­
tions have not been reported.
This study was designed to determine if immunosuppression results
from BVD infection and, if so, how it would affect the recovery from a
concurrent viral infection.
Infectious bovine rhinotracheitis (IBR) virus
was selected to measure this effect, since it is frequently associated
with BVD virus in disease conditions of feedlot cattle.
Combined BVD-IBR
infections are a common finding when groups of cattle from different
origins are assembled in feeder operations in Iowa and elsewhere, usually
resulting in severe economic losses.
Enhanced IBR virus multiplication,
often with generalized infection, is a finding cotmon to both natural
outbreaks and postvaccinal reactions.
If infection with BVD virus inter­
feres with resistance to IBR virus infection, the severity of the clinical
disease could be significantly enhanced.
The objectives of the experimentation reported in this dissertation
were:
(1) to study the normal process of recovery from IBR and BVD virus
infections by analyzing the specific and nonspecific immunologic re­
sponses, with emphasis on cellular immunity; and (2) the effect of con­
current BVD virus infection on the process of recovery from IBR virus
infection.
3
REVIEW OF LITERATURE
Resistance to Viral Infection
The defense mechanisms that enable man and animals to resist a viral
infection, or to recover from it, comprise both specific and nonspecific
factors.
The nonspecific mechanisms, usually active against a variety of
viruses, constitute what Raffel (1961) termed "native immunity", and are
for the most part a genetic property of an animal species.
The specific
inrnune mechanisms, on the contrary, are more of an individual property.
They are dependent upon the individual's exposure to a particular antigen
and, on a purely mechanistic basis, the types of responses tend to spread
through broad evolutionary steps rather than being confined to particular
animal species.
Nonspecific factors
The obligatory intracellular nature of parasitism by viruses requires
the presence of susceptible tissues in an animal for virus multiplication
tc t2K2 place.
The absence c-f ^"rn hissu^s, confers a ccndition of total
resistance to the particular agent irrespective of infectious dose or
general resistance of ths host to infectious diseases (Smorodintsev,
1960).
However, even potentially pathogenic viruses can fail to gain
entrance into the organism or, even if initial virus replication takes
place, the host can achieve extinction of the agent by the role of a
series of nonspecific factors.
Epithelial surfaces — the skin and mucous membranes
The epithe­
lial surfaces provided by the skin and mucous membranes offer a physical
barrier to penetration by viruses.
In addition to this passive effect,
4
lactic acid, fatty acids and lysozyme present in sweat and sebaceous se­
cretions confer bactericidal and viricidal properties (Raffel, 1961).
Similar factors exist in the moist mucous films of the respiratory and
genital mucosas, and also in the saliva and gastrointestinal secretions
where pH changes and strong enzymatic activity offer added protection.
The indigenous microbial flora, particularly at the level of the
gastrointestinal tract plays a very important role in host resistance to
infectious diseases (Abrams. 1970).
Physiological and morphological
parameters of the small intestine with particular reference to enzymatic
activity, intestinal absorption, epithelial cell renewal and lymphoid
development, have been shown to be directly related to the presence of
intestinal flora as. observed in comparison of conventional and germ free
animals.
The ciliary movement of the respiratory epithelium is very effective
in cleaning mucus and particulate materials from the airways and is prob­
ably an important factor in resistance to respiratory infections.
How­
ever, airborne viruses c?n nrnhflnly rmAch t.liè lower respiratory tract by=
passing the clearance mechanism, when sufficiently small particles are
present in a viral aerosol (Knight, 1973).
In addition, viruses such as
influenza and parainfluenza in man have been shown to have specific af­
finity for cilia, attaching firmly to them and possibly contributing to
cell infection (Tyrrell, 1973).
Virus neutralizing factors in blood and body tissues
A number of
substances in normal serum, body fluids and tissues are capable of neu­
tralizing a wide range of viruses, and are very likely to be of signifi­
cance in the host resistance to viral infections.
The bactericidal
5
enzyme, lysozyme, recognized by Fleming in 1922, occurs in tissues and
several body fluids (Raffsi, 1961).
by Ferrari et al. (1959).
Its antiviral activity was recognized
A variety of thermolabile, nonspecific inhib­
itors are found in the serum and tissues of man and a variety of warm
blooded animals (Raffel, 1961; Smorodintsev, 1960; Nash et al., 1971).
They can inhibit the infective or hemagglutinating activity of a wide
range of viruses, including all the myxoviruses, many arboviruses, adeno­
viruses and enteroviruses (Ginsberg and Horsfall, 1949; Porterfield and
Rowe, 1960).
Many of these inhibitors seem to be lipidic in nature
(Smorodintsev, 1960; Porterfield and Rowe, 1960; Salminen, 1362;
Sanderson, 1968; Bidwell and Mills, 1968).
Phosphatase enzymes of tissues
have been shown to neutralize herpes simplex virus (Amos, 1953).
A complement-requiring plasma protein possessing bactericidal proper­
ties, properdin, was described by Pillemer et al. in 1954.
Ginsberg and
Wedgwood (1960) ascribed virus neutralizing properties to this protein,
with action on Newcastle, influenza, T-bacteriophages and other viruses.
Although thc:c proteins v.'sre disn^issed by ni?.ny ?? n^furHi rtnf.inoiilHs it
has been shown recently (Pensky et al., 1968) that it is indeed a unique
serum protein, nonimmunoglobulin in character, with the capability of
cleaving the C3 complement fraction (alternative pathway for complement
activation) (Lepow, 1971).
Body temperature
Smorodintsev's experiments (reviewed by
Smorodintsev, 1960) showed that a number of animal viruses such as in­
fluenza, Japanese encephalitis, ectromelia and vaccinia are inactivated at
normal body temperatures.
Observations that an increase in body tempera­
ture usually results in faster rates of inactivation of the virus or
6
increased survival of susceptible animals, are far more significant from
the standpoint of resistance to infection since they imply that febrile
responses can be a major factor in the recovery process.
Smorodintsev
(1960) reported that increases of body temperature of both susceptible and
insusceptible animals resulted in a faster rate of inactivation of in­
fluenza virus.
Walker and Boring (1958) observed that the multiplication
of Coxsackie virus and its pathogenicity for mice depended upon body
temperatures; artificially lowered temperatures resulted in increased
lethality, increased temperatures resulted in failure of virus multiplica­
tion and complete survival.
Similar results were reported by Lwoff (1959)
with poliovirus in mice.
The preceding observations suggest that fever can be an important
factor in resistance of the host to the spread of viruses in the organism.
The mechanism of action is unknown, however, and, since fever results in
many secondary changes in metabolic activity, it is possible that the in­
creased temperature per se is not the main factor in viral inactivation.
Lwuff (1953) has l,ypùuucsizcd tliat the high temperature result: In release
of lysosomal nucleases responsible for destruction of viral RNA during
virus replication.
Inflammatory reaction
Although there is no doubt about the value
of local inflammation in the native defense of the host during bacterial
infections, it may be of lesser importance in resistance to viral infec­
tions.
The inflammation that accompanies viral infections is usually
secondary to the primary cellular alterations and the polymorphonuclear
response is usually lacking (Bruner and Gillespie, 1973).
Nonetheless,
the increase in temperature, localized acidity, decreased oxygen tension,
7
changes in osmolarity and salt concentration, and local exudation, could
be factors in inactivation or localization of many viruses (Baron, 1973).
Phagocytosis
Although phagocytic cells constitute a very im­
portant factor among the defense mechanisms of the body due to their
ability to ingest and dispose of foreign material, their role in viral
infections is highly variable.
Virus particles can be taken up by phago­
cytes in the same manner as nonviral particles.
Blood clearance studies
of viral particles in mice have shown that the rate of clearance depends
largely on the size of the particles.
Small viruses such as T7 bacterio­
phage and Rift Valley fever virus are taken up much more slowly than
larger viruses such as vesicular stomatitis virus or vaccinia virus
(Mims, 1964).
Phagocytosis of a virus by macrophages does not necessarily imply an
effective defense mechanism.
Viruses such as vaccinia, influenza and
myxoma in the mouse, aid ectromelia in the rat, are cleared by liver
macrophages, digested and degraded.
Other viruses such as distemper virus
in tluyb, yellow fcVci" virus in mcr.kcys sr.ci cctrcmslia in the mouse; r*,,
replicate in macrophages and later spread to other susceptible cells.
These observations suggest that, at least in some cases, the final outcome
of an infection could largely depend on the initial virus-macrophage
interactions (Mims, 1964).
Available Information strongly Indicates that resistance to some
virus infections is expressed through macrophages.
Although both adult
and suckling mice are susceptible to encephalitis induced by intracerebral
Inoculation of herpes simplex virus, only suckling mice develop encepha­
litis after peripheral inoculation.
Johnson (1964a) observed that the
8
initial replication of herpes simplex virus after extraneural inoculation
of suckling mice takes place in macrophages including subcutaneous or
submucosal histiocytes, alveolar macrophages or peritoneal macrophages de­
pending upon the route of inoculation.
The virus later spreads to other
tissues and to the CNS via neural or hematogenous routes.
In contrast,
the infection is arrested at the macrophage level in adult mice.
In vitro
experiments have shown that although the virus replicates equally well in
macrophages from suckling or adult mice, the latter have the ability to
restrict its spread to contiguous cells (Johnson, 1964b).
of this action has not been described.
The mechanism
Similar observations were reported
by Gal lily et al. (1967) with mouse hepatitis virus and macrophages from
CH3 mice.
Genetic resistance of some strains of mice to many arboviruses
is also related to the ability of macrophages to destroy the virus
(Goodman and Koprowsky, 1962a,b; Olson et al., 1975).
The cited examples stress the possible significance of the macrophage
in natural resistance to viral infections, but macrophages also play a
very impur târiL rolè 1,1 Specifically Gcquirsd iïïr.ur,ity.
In addition to
their purely phagocytic and virus destroying properties, their participa­
tion in the early stages of the immune response can be subject to genetic
variation.
In this regard, Belgrade and Osborn (1974) have reported that
in strains of mice with different degrees of susceptibility to murine
cytomegalovirus infection, the difference in susceptibility can, to a
certain degree, be due to differences in macrophage function during the
inductive phase of the iimune response.
Interferon
Interferon was first described by Isaacs and Lindenmann
(1957) as a cell protein, produced in response to viral infection, with
9
the property of conferring on cells resistance to the multiplication of
viruses.
A modern definition would be:
"Interferons are cell coded pro­
teins induced by foreign nucleic acids; they are nontoxic for cells but
are able to inhibit the multiplication of vertebrate viruses in cells of
homologous species" (Fenner et al., 1974).
Interferon is today considered a family of proteins since their
molecular weights vary considerably depending upon the source.
varies from 28,000 to 160,000 (Friedman and Sonnabend, 1970).
The range
All the
interferons so far examined are nondialyzable, not sedimented by centrifugation at 100,000 g for 4 hrs, susceptible to proteolytic enzymes, and
are stable over a wide pH range (2 to 10).
A possible exception to this
pH resistance is the interferon produced in leukocytes in response to
phytohemagglutinin (Wheelock, 1965), which was stable at pH 3-9 but not at
pH 2 or 10.
In an appropriate host, virtually all animal viruses induce formation
of interferon.
Double stranded RNA (viral RNA, replicative intermediates,
or suiiie uuuule buatiuêu RNA Cùniplcxes observed during normal transcription
of DNA viruses) seems to be the inducer molecule (Colby and Morgan, 1971).
The mechanisms of interferon induction and action are still largely
unknown,
A derepression hypothesis of interferon synthesis (Burke, 1966)
is widely accepted but has not been proven.
It suggests derepression of
the cell genome by the viral inducer resulting in synthesis of interferon
and any other proteins involved in its production.
According to Marcus
and Salb (1966) interferon would then act on other cells by inducing the
synthesis of a cell coded "translation inhibitory protein".
This protein
would bind to and modify the ribosomes capacity to translate viral RNA.
10
These specific defects in the function of ribosomes were not confirmed
however in the detailed analysis of Kerr et al. (1970).
Recent evidence
implicates transcription of viral RNA as the basic defect in the viral
replication mechanism (Bialy and Colby, 1972).
Whatever its mechanism of action, interferon produced by one cell
type is active against a wide variety of viruses although the sensitivity
of different viruses varies markedly (Stewart et al., 1969).
Interferon
has a high degree of host-cell species specificity although it shows no
evidence of organ or tissue specificity (Fermer et al., 1974).
Specifically acquired immunity
The specific immune responses are concerned with recognition of the
foreign nature of sustances, microorganisms or cells introduced into the
body, and the subsequent reaction of the host toward these foreign ma­
terials.
This reaction encompasses a series of cellular interactions ex­
pressed by the elaboration of specific cell products and by specific cell
changes in morphology, function or both.
Bellanti (1972) established
these yerier-dl uharàuLet isl'ics ûf the specific iiTuT.LiriS response v;h1ch dis­
tinguishes it from the nonspecific response;
(1) specificity of the re­
actions toward the inducing agents; (2) heterogeneity of the cell types
and cell products involved and their mechanisms of action; and (3) memory ;
a property which results in augmentation of the response through prolifer­
ation and differentiation of cells upon subsequent exposure to the origi­
nal inducer.
There are two general types of effector mechanisms which mediate
specifically acquired immunity, those mediated by cell products of the
n
lymphoid tissues (antibodies, humoral immunity) and those mediated by
lymphocytes with or without cooperation of other cell types (cell-mediated
immunity).
These effector mechanisms evolve from two functionally different
lymphoid systems, one developing under thymic influence and another de­
veloping under an alternative influence which has been precisely located
only in birds in the bursa of Fabricius.
The concept of these dual iimmu­
nity systems evolved from tissue extirpation experiments and clinical
observations.
Glick et al. (1956) observed that removal of the bursa of
Fabricius from newly hatched chickens resulted in impaired capacity to
produce antibodies.
Aspinall et al. (1963) reported retarded homograft
rejection in chickens after neonatal thymectomy.
Cooper et al. (1966}
provided definitive evidence for a dichotomous immune system in chickens,
in which the bursa of Fabricius controlled antibody formation and the
thymus cell-mediated reactions.
Similar observations were reported in rodents (Good et al., 1962)
where thymectomy also resulted in impaired cell-mediated iimiune reatliuny
and lymphopenia.
Lymphocyte depletion resulted only in certain areas of
the lymphoid tissues (thymus-dspendent areas), while follicles, germinal
centers and plasma cells are unaffected (Waksman et al., 1962).
A series
of clinical observations in man by Good and his colleagues (reviewed by
Good et al., 1971) on immune deficiency diseases such as Burton agairanaglobulinemia, Di George's syndrome, thymomas and myelomas, further con­
firmed the existence of a dual immune system in mammals.
However, the
mammalian equivalent of the bursa of Fabricius has not been definitely
identified.
12
The functionally distinct lymphocytes were designated "T" cells (for
thymus-derived or thymus-dependent) and "B" cells (for bursa or bursaequivalent-derived).
These two cell types possess differential charac­
teristics such as presence or absence of surface immunoglobulins, antigens
or receptors, tissue distribution, function, response to mitogens and
susceptibility to X-irradiation, corticosteroids or antilymphocyte serum
(Greaves et al., 1973).
They are not homogeneous populations, however,
and functional subpopulations have been described (Greaves et al., 1973).
It must be emphasized that the functional separation of T and B cells
is not absolute, and that cell interactions (lymphocyte-lymphocyte and
macrophage-lymphocyte) are very important at all stages of immune re­
sponses.
Greaves et al. (1973) have reviewed lymphocyte-lymphocyte inter­
actions and divided them into three general categories:
(1) B-B and B-T interactions -- B cells can influence the response of
other B cells through the action of antibody, and "feedback" regulation of
antibody response by antibody was demonstrated in several systems (re­
viewed by Uhr and moller, 1966).
responses.
Likewise, antibody can ir.fluSr.cc T call
Kaliss (1959) reported enhancement of tumour growth by passive
antibody, and Axelrad (1958) reported partial inhibition of delayed hyper­
sensitivity to sheep red blood cells in rats by passively injected anti­
body.
Conversely, Liew and Parish (1972) reported suppression of antibody
responses to flagellin and sheep erythrocytes in rats by passive antibody,
with accompanying enhanced cell-mediated immunity.
(2) T-B cell cooperation — The existence of T-B cell interaction in
antibody production has been demonstrated beyond any doubt in many ex­
perimental systems and for a variety of antigens (reviewed by Greaves
13
et al., 1973).
After the original observation of Ovary and Benacerraff
(1963) that secondary responses to hapten-carrier complexes was only
possible if the same carrier was used in both inoculations, continued
work on the subject finally led to the conclusion that T cells collaborate
in the immune response by recognizing "helper" determinants in the car­
rier molecule, somehow "helping" B cells to make antibody to the haptenic
antigenic determinants (Rajewsky et al., 1969).
action is still obscure.
The nature of the inter­
It could involve (a) antigen bridges (Mitchison
et al., 1970) between T and B cells or T cells, B cells and macrophages;
(b) release of short range factors by T cells (active on B cells or B
cells and macrophages) either antigen specific (Feldmann et al., 1973) or
nonspecific (Button et al., 1971); or (c) a combination of both.
(3) T-T cell cooperation — Different T cell subpopulations may
cooperate in graft-versus host responses as described by Cantor and
Asofsky (1972) but the nature of those interations is still unclear.
Macrophage-lymphocyte interactions are also very important in both
inductive ana effector stages of specific iiiiiiiuiic respGnses (Gottlieb and
Waldman, 1972; Nossal and Ada, 1971; Unanue, 1972; Schwartz et al., 1970).
Macrophage-depleted lymphocyte cultures give only a weak proliferative
response to soluble antigens or allogeneic cells (Oppenheim et al., 1968)
and marginal or negative antibody responses to most antigens (Feldmann,
1972).
Macrophage-bound antigen constitutes a very effective trigger for
both T and B lymphocytes (Katz and Unanue, 1973).
After antigens are
broken down by macrophages a persistent fraction is found on the cell
membrane (Unanue and Cerottinij 1970).
These cell surface associated
antigens are probably essential for the macrophage-lymphocyte interactions
14
in the induction of the immune response, considered to be a surface
phenomenon involving antigen presentation (Unanue, 1972).
Macrophages act
nonspecifically (Greaves et al., 1973) and they display a high level of
activity, even when present at very low concentration (under 1%) in a
given population (Rosenstreich and Rosenthal, 1974).
Humoral antibody response to viral infections
The replication of
viruses in the host produces several different antigens.
Some are struc­
tural components, but many of them are virus induced enzymes.
In poxvirus
infected cells, less than one-half of the antigens detected are incorpo­
rated into the virions.
Probably only a few of the antibodies which react
with these viral antigens play a significant role in protection (Fenner
et al., 1974).
Five main classes of immunoglobulins have been identified in man:
IgG, IgM, IgA, IgD and IgE (Syîêgelberg, 1974).
Of these, at least three
(IgG, IgM and IgA) contain antiviral antibody activity (Bellanti and
Svehag, 1971).
These three immunoglobulins constitute by far the bulk of
the immunoglobulin prebenL in liuman seriiir. with the ccrr.bir.ed average con­
centration about 18 mg/ml compared to 0.03 mg/ml for IgD and IgE combined
(Spiegelberg, 1974).
IgM and IgG (îgGi and IgG, and somewhat less IgG?)
can fix complement.
IgA can fix complement only by the alternate or by­
pass mechanism.
IgG (IgGi and IgGg) can bind to macrophages (Spiegelberg,
1974).
Analogs of three of those classes (IgG, IgM and IgA) have been
identified in the bovine species (Porter, 1973; Butler, 1973).
Homo-
cytotropic antibody has also been reported in what apparently is a dis­
tinctly new immunoglobulin class (Hammer et si., 1971).
The IgG^ subclass
15
of bovine IgG fixes complement (Porter, 1973), but the bovine IgM is a
weak complement fixer (Rice, 1968).
At least the IgG and IgM classes of
serum antibody display antiviral activity (Cowan and Wagner, 1971).
In general, IgM antibodies appear earlier in the serum than IgG
antibodies (Rice, 1968).
This sequential appearance of the different
classes of antibodies has been observed both after natural viral infection
or inoculation with live vaccinal strains (Ogra et al., 1958; Bellanti and
Artenstein, 1954), and after parenteral inoculation of nonreplieating
viral antigens (Svehag and Mandel, 1964a,b; Svehag, 1964a,b; Webster,
1965, 1968a,b).
Webster's experiin^nts, utilizing influenza virus antigens
in rabbits, showed that the IgM antibodies were of high avidity and with
high cross reactivity while the initial IgG response was of low avidity
and greater specificity.
The avidity of these IgG antibodies increased
with time.
The basis for inhibition of virus infectivity due to neutralization
by antibody has not been adequately established.
It has been shown re­
peatedly InàL viruses are not rendered Irrsvsrsib'.y noninfectinus hy
neutralizing antibody (Mandel, 1971).
Early views of the neutralization
phenomenon considered antibody as inhibiting attachment of virus to the
cell and preventing engulfment of attached virions (razekas de St. Groth,
1962).
However, there is now evidence to the contrary.
Smith et al.
(1961) found that although antiserum to vaccinia virus reduced plaque
formation in L cells 100%, 20% of the particles were attached to the cells
but still failed to initiate infection.
Dales and Kajioka (1964) reported
that vaccinia virus coated by antibody was still able to attach to L
cells, and eventually be engulfed.
However s antibody reduced the number
of cell-associated virions, blocked the release of virus from phagocytic
vesicles and rendered the virus susceptible to degradation within the
vesicle.
Silverstein and Marcus (1964) reported similar results for
Newcastle disease virus.
Mandel (1967a,b) found that most of neutralized poliovirus attached
strongly to HeLa cells, but its viral RNA was completely degraded by the
cells.
The finding led to the suggestion that antibody brings about a
change in the normal mode of attachment of poliovirus with the viral
particles being actively phagocytized and brought into contact with
lysosomes.
It should be emphasized that the neutralization process and its
efficiency not only depends on the antibody, but also on the particular
cells used as an indicator (Lafferty, 1965).
While chicken antibody
actually enhanced the infectivity of flaviviruses for chick embryo fibro­
blasts, it neutralized their infectivity for heterologous cells (Hawkes
and Lafferty, 1967).
ihs prcccss of virus neutralization is nmhanly nmrA effective in
vivo due to the action of complement.
Berry and Almeida (1968) showed
that avian bronchitis virus is actually destroyed by complement after re­
action with antibody in a mechanism resembling the lysis of erythrocytes
by IgM antibody and complement.
Complement greatly enhances the neutral­
izing capacity of IgM and early IgG antibody, although it usually does not
affect late IgG antibody (Hampar et al., 1968; Potgieter, 1974).
Early
whole antiserum is also dependent on complement for optimal neutralization
(Yoshino and Taniguchi, 1969; Potgieter, 1974).
17
The mechanism of action of complement is not well understood.
Wallis
and Mel nick (1971) proposed that early antibodies are monovalent and fail
to aggregate the virus without complement.
immunoaggregation.
Complement would produce
The reason for the difference between early end late
IgG antibodies in complement dependency probably lies in their differences
in specificity, affinity, valence and configuration (Blank et al., 1972).
Still another possible mechanism of antiviral activity under in vivo
circumstances is the promotion of phagocytosis by opsonizing antibodies
(Mims, 1964).
Cell-mediated immune response to viral infections
There is now
abundant evidence that viruses elicit cell-mediated iiraiunity.
Initial
interest in this field was derived mainly from clinical observations in
man, where a number of conditions were associated with enhanced suscepti­
bility to viral infections (Glasgow, 1970; Allison and Burns, 1971).
The
common denominator of these conditions was impairment of at least one
component of the immune response, and particularly, thymic deficiency.
Patients wiLh uncomplicated ssvsrc hypogsirr^gicbiillp.eir^ia rcccvcrcdrorzsTly
from measles, varicella, or small pox vaccination.
However, in patients
with congenital abnormalities predominantly affecting cell-mediated immu­
nity such as ataxia-telangiectasia, Swiss-type agammaglobulinemia, and the
syndromes of Wiskott-Aldrich, Gitlin, Nezelof and Di George, virus in­
fections were a recurring problem.
Cancer chemotherapy, immunosuppressive
therapy after tissue transplants, and Hodgkin's disease, which also de­
press cell-mediated immunity, result in increased susceptibility to viral
infection.
Cytomegalovirus, herpes simplex, varicella and vaccinia
18
viruses are some of the most common agents involved (World Health Organi­
zation, 1973).
The preceding observations were paralleled by experimental infections
in which host resistance had been altered by various immunosuppressive
regimens.
Glasgow (1970), Allison (1972b) and North (1974) have reviewed
some of this work.
The general observation has been of increased suscep­
tibility in laboratory animals to viruses such as herpes simplex, ectromelia, vaccinia or oncogenic viruses such as polyoma or leukemia viruses
after treatments principally affecting cell-mediated immunity (thymectomy,
antilymphocyte serum).
A common pattern has emerged from both clinical and experimental
work; viruses which can express viral antigens in the host's cell membrane
(i.e., poxviruses, herpesviruses, oncogenic viruses) elicit cell-mediated
immune responses, and these responses seem to be essential for host re­
sistance.
"with virus infections where plasma viremia is the rule and cell
membrane modifications are not prominent (i.e., enteroviruses, togaviruses), cell-rued id Leu iiimiuii i ty is usually "Gt elicitsd and circulating
antibody seems to play a preponderant role in host resistance to infection
(World Health Organization, 1973; Allison; 1971),
For years, the skin delayed hypersensitivity reaction was the only
method of testing for cell-mediated immune responses in the live animal.
But histological studies of the skin reactions and the passive transfer
of dermal reactivity with lymphoid cells, resulted in the identification
of the specifically sensitized lymphocyte as the cell responsible for this
reaction.
Subsequently, the study of the interactions in vitro between
antigens and sensitized lymphocytes, and the apparent correlation between
19
some of these interactions and the condition of delayed hypersensitivity,
led to the establishment of a series of in vitro correlates of cellmediated immunity.
These correlates have been extensively detailed or
reviewed by Lawrence and Landy (1969), Bloom and Glade (1971), Revillard
(1971), World Health Organization (1973) and McCluskey and Cohen (1974).
Therefore, consideration of this subject will be limited with emphasis on
those mechanisms and applications that relate more directly to the ex­
perimentation described in this dissertation.
Lymphocyte stimulation
Lymphocytes cultured in vitro and
exposed to a variety of stimulants respond with morphological enlargement
to large lymphoblasts (large cells having a basophilic cytoplasma and one
or more nucleoli) and by cell proliferation (Robbins, 1964; Oppenheim,
1968; Valentine, 1971).
This cell proliferation and transformation can be
quantitated by lymphoblast counts in stained preparations, or much more
accurately by the measure of tritiated thymidine uptake, an indicator of
total ONA synthesis, or uptake of radioactive aminoacids, an indicator of
•
•
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jjr uueni bytii-neb ib
r%t . r \ t
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.
_
aiiu vuiicii, is/t/.
Lymphocytes can be stimulated by specific antigens or by nonspecific
stimulants which do not need prior sensitization of cell donors to be
effective (Oppenheim, 1968).
Phytohemagglutinin (PHA), pokeweed mitogen
(PWM), concanavalin A (Con A) and bacterial lipopolysaccharides (LPS) are
among the most widely employed nonspecific stimulants (Hirschhorn and
Hirschhorn, 1974).
PHA and Con A stimulate T cells in man and mouse
(Janossy and Greaves, 1971; Anderson et al., 1972; Mellstedt, 1975).
PWM
stimulates both T and B lymphocytes (Douglas, 1972; Janossy and Greaves,
1972) at the usual concentration, although at low concentration it seems
20
to stimulate B cells only (Ivanyi and Lehner, 1974).
LPS is a B cell
stimulant (Ivanyi and Lehner, 1974; Peavy et al., 1974).
A somewhat
different group of nonspecific stimulants would include anti-gammaglobulin, selectively stimulating B cells, which bear immunoglobulin receptors
(Hirschhorn and Hirschhorn, 1974).
The specific-antigen stimulants require previous lymphocyte sensiti­
zation of the donor.
response.
The stimulation thus constitutes a specific iirsnune
This implies a recognition step by specific receptors on
lymphocyte membranes.
While B lymphocytes have detectable membrane
immunoglobulin receptors that are capable of binding the antigen for
which the cell is specific (Davie and Paul, 1971; Davie et al., 1971),
there is no agreement on the nature of the receptors on T lymphocytes.
Some workers detected immunoglobulins on the membrane of T lymphocytes
(Marchalonis et al., 1972; Hammering and Rajewsky, 1971) whereas other
workers have failed to do so (Vitetta et al., 1972; Perkins et al., 1972).
Waldron et al. (1973) clearly demonstrated the requirement for macro­
phages for the in vitro stimulation of sensitized guinea pig lymphocytes
by PPD.
They concluded that macrophages play an obligatory role in the
proliferative response to specific mitogens (Waldron et al.. 1973;
Waldron et al., 1974).
A number of other workers have reported this need
for an interaction between macrophages and T lymphocytes for optimal
stimulation of lymphocytes by antigen (Cline and Swett, 1968; Hersh and
Harris, 1968; Seegh and Oppenheim, 1970; Cohen and Paul, 1974; Lipsky and
Rosenthal, 1975).
Cytophilic antibody on the surface of the macrophage is
a possible mechanism for mediating the reaction by contributing to antigen
binding (Cohen et al., 1973; Cohen and Paul, 1974).
However, Rosenthal
21
and Shevach's observation (1973) that identity between histocompatibility
antigens of macrophages and lymphocytes is necessary for antigen presenta­
tion suggests a more complex system than simply antigen specific immuno­
globulin receptors on the effector cells.
A third category of stimulants that does not really fit either of
the preceding classes includes the allogeneic lymphocytes and the antiallotypic antibodies.
Cocultivation of lymphocytes (mixed lymphocyte
culture) from genetically unrelated individuals results in cross stimula­
tion, referred to as the mixed lymphocyte response (MLR).
This reaction
resembles nonspecific stimulation in that there is no need for prior
sensitization, but it also resembles specific stimulation because recog­
nition of antigenic difference is involved (Hirschhorn and Hirschhorn,
1974).
The nonspecific stimulants find clinical application in evaluation of
lymphocyte reactivity for diagnosis of immunodeficiency diseases (con­
genital or acquired) or for evaluation of immunosuppressive treatments.
They also have been extensively used in experimetiLaLioii fw, devclcpmsr.t
and evaluation of immunosuppressive drugs, studies on immunomanipulation
and study of the pathogenesis of some viral diseases (Revillard-, 1971).
Specific stimulation of lymphocytes by viral antigens has been used
in a number of viral diseases to evaluate cell-mediated iiraiune responses.
Included in these studies are the following:
herpes simplex virus
(Rosenberg et al., 1972; Wilton et al., 1972; Starr et al., 1975); varicella-zoster virus (Jordan and Merigan, 1974); vaccinia virus (Rosenberg
et al., 1972; Elves et al., 1953); rubella virus (Lee and Sigel, 1974;
Morag et al., 1974); measles virus (Graziano et al., 1975); Sindbis virus
22
(Griffin and Johnson, 1973); mumps virus (Smith et al., 1972); EpsteinBarr virus (Gerber and Lucas, 1972); rabies virus (Wiktor et al,, 1974;
Hill, 1974) and infectious bovine rhinotracheitis (Davies and Carmichael,
1973; Rouse and Babiuk, 1974a).
In most of the studies there was evidence
that the period of lymphocyte reactivity is short-lived; reactivity is
first detected 3-5 days after inoculation, usually peaks at approximately
1 week post inoculation and drops to or near background levels in 2-3
weeks post inoculation (Rosenberg et al., 1972; Griffin and Johnson, 1973;
Davies and Carmichael, 1973; Rouse and Babiuk, 1974a; Hill, 1974).
The
degree of stimulation can be enhanced by purificati i of the virus prep­
arations, although relatively crude viral antigens usually provide satis­
factory stimulation (Rosenberg et al., 1972).
Inactivated virus should be
used as the source of viral antigen, especially for those viruses able to
multiply in macrophages or lymphocytes, since viral multiplication can
result in depression of cell metabolism (Rosenberg et al., 1972).
Cytotoxic reactions
Cytotoxic effects of lymphocytes on
adjacent cells were first clearly ubberveu iii Iristological secticr.s of
tumors undergoing immunological rejection and subsequently recognized in
allografts of normal tissues, in contact allergy and in a variety of
experimental autoallergic lesions (Waksman, 1974).
A variety of experimental techniques have been developed to charac­
terize the cytotoxic reaction and describe the mechanisms of target cell
destruction.
Several distinct types of in vitro cytotoxic mechanisms have
been recognized (Perlmann and Holm, 1969; Waksman, 1974);
(1) Direct cytotoxic effect on target cells by lymphocytes from
sensitized donors -- These reactions require adherence of the sensitized
23
lymphocytes to the target cells, an immunologically specific event, while
the subsequent target cell effects are nonspecific (Wilson, 1965a,b).
Antibody and complement play no role in the process (Rosenau and Moon,
1961).
The specific receptors on the effector lymphocytes recognize the
responsible antigens on the cells.
These can be natural components
(Dumonde, 1966) or artificially attached antigens (Perlmann et al., 1970).
This initial reaction triggers the subsequent killing of the target cells.
The lymphocytes responsible for this direct cytotoxicity are T cells
(Brunner and Cerottini, 1971; World Health Organization, 1973; Wilson,
1963).
The killing of target cells does not require blast transformation
(Shaks and Granger, 1971; Lundgren and Moller, 1969) and is not mediated
by released cytotoxic factors (Canty and Wunderlich, 1970).
No more than
1 to 2% of a population of lymphocytes are active (Wilson, 1965a) and,
accordingly, a lymphocyte-target cell ratio near 100:1 is usually required
to obtain peak activity.
The nature of the cytotoxic reaction remains unclear although the
effect seems to be primarily on cell membranes (Able et al., iy7u)-
A
passage of DNA and RNA from lymphocytes to target cells has been reported
but the action on the target cell is unknown (Waksman, 1974).
The techniques used for measuring cytotoxicity have been extensively
reviewed and evaluated by Perlmann and Holm (1969).
These techniques in­
clude simple morphological observation, counting of surviving cells or
nuclei, production of plaques of cell lysis, use of trypan blue to esti­
mate percentages of dead cells, inhibition of cloning or colony formation,
release of labeled nucleic acids or proteins, and release of sicr.
24
The latter technique was first utilized for studies on the survival of red
blood cells in hemolytic diseases and has been widely employed.
It was
found to provide an excellent label for determination of cell-mediated
lysis of tissue culture cells (Holm and Perlmann, 1967), and of chicken
erythrocytes (Perlmann et al., 1968).
s^Cr is noncovalently bound to
proteins and other cell constituents.
The chromate is reduced during
binding and isotope is not reutilized (Holm and Perlmann, 1967).
Labeling
is rather stable in chicken erythrocytes, and the technique represents a
sensitive index of cell damage (Perlmann et al., 1968).
A wide variety of cells have been used as target cells in direct
cytotoxicity systems.
Tumor cells and established cell lines have been
employed most extensively but many studies have utilized virus infected
cells as target cells to evaluate antiviral immunity.
Speel et al. (1968)
reported a cytotoxic effect of spleen cells from mumps virus-immune mice
on a human cell line persistently infected by mumps virus.
Cytotoxicity
was determined by visual determination of the percentage of dead target
cells.
Lundstedt (i959) used a similar technique to deLennint: ^.ylotoxic
activity of itrenune mouse spleen cells for LCM virus-infected L cells.
The
chromium release assay was used to detect immune cytotoxic activity of
human lymphocytes against mumps virus-infected Vero cells (Andersson
et al., 1975), herpes simplex-infected human amnion cells (Russell et al.,
1975); BHK-21 cells persistently infected with rubella virus (Steele
et al., 1973, 1974) and HeLa and WI-38 cells persistently infected with
measles virus (Labowskie et al., 1974).
Wilton et al. (1972) used siCr-
labeled chicken erythrocytes as a nonspecific indicator of cytotoxicity
by human lymphocytes previously stimulated by herpes simplex virus
25
(specific stimulation).
Gardner et al. (1974a) reported specific release
of siCr from ectromelia-infected mouse embryo cells, chicken embryo cells,
mouse fibroblasts and mastocytoma cells when incubated with spleen cells
from ectromelia immune mice.
McFarland (1974a) reported specific cyto­
toxicity of Sindbis virus-infected mouse embryo cells by Sindbis virussensitized mouse spleen cells.
He utilized release of ^^sj.-jgdoeoxy-
uridine to measure the cytotoxicity.
Several of the above mentioned studies (Gardner et al., 1974a,b;
McFarland, 1974a; Andersson et al., 1975) demonstrated that T lymphocytes
were responsible for the cytotoxic effect.
In addition, McFarland (1974a)
reported that macrophages were responsible for clearing the virus released
from the lysed cells and suggested that the final "recovery" in his iji
vitro model of an acute virus infection depends on a balance between
lymphocytes and macrophages.
(2) Release of soluble cytotoxic mediators by sensitized lymphocytes
-- The production of cytotoxic mediators by sensitized rat lymphocytes
reacting witn specific antigen was first desuribeu uy Ruclcllè and naksmari
(1968).
Granger and his coworkers (Granger and Williams, 1968; Kolb and
Granger, 1958; Granger et al., 1959) made a similar observation relative
to mouse and human lymphocytes after stimulation by PHA, histocompati­
bility antigens and specific antigens.
toxin".
They introduced the term "lympho-
Production of lymphotoxin requires the presence of macrophages,
but it is produced by T lymphocytes (Waksman, 1974).
Lymphotoxin is non­
specific in action and is capable of lysing a whole variety of cell types
including cells from different animal species.
Different cells, however,
exhibit different degrees of sensitivity to lysis by a single preparation
26
of lymphotoxin (Granger and Williams, 1971).
The site of action of
lymphotoxin seems to be the cell plasma membrane (Granger and Williams,
1971).
Kanra and Vesikari (1975) recently reported production of a lymphotoxin-like mediator by lymphocyte cultures from rubella-sero positive
human donors in response to stimulation by inactivated rubella virus.
This preparation showed preferential cytotoxicity for rubella-infected
target cells in comparison with uninfected cells.
Therefore, more inves­
tigation is needed before this reaction can be considered true lymphotoxin
production.
(3) Cytotoxicity mediated by antibody to target cells and normal
lymphocytes — Antigenic target cells, treated with certain heat inacti­
vated antisera will be damaged when exposed to lymphoid cells from unsensitized donors.
The antisera are effective at dilutions too high to
give rise to conventional complement-mediated lysis in the absence of
lymphoid cells (Perlmann and Holm, 1969).
The antibody responsible for
this activity belongs to the îyG uidbb anu ail fûur siibclôsscs of
IgG are active in this regard (MacLennan and Howard, 1972; MacLennan
et al., 1970).
There is lack of agreement on the type of lymphoid cell responsible
for ihis type of cytotoxicity.
Cells are activated through Fc receptors;
modified Fc fragments of IgG bound to antigens on the surface of the
target cell will bind to these receptors and activate the cells (Allison,
1972a).
The effector cell is not phagocytic, requires close contact with
the target cell and kills by surface contact (Perlmann and Perlmann, 1970;
Allison, 1972a).
Early investigators considered the effector cells to beB
27
lymphocytes (MacLennan and Harding, 1970; Van Boxel et al., 1972), but
later experimentation with extensively purified cell preparations demon­
strated that they are neither T cells nor immunoglobulin-bearing B cells
(Greenberg et al., 1973a).
Greenberg et al. (1973b) and Allison (1972a)
considered these cells to be nonphagocytic monocytes in view of the glassadherent properties they observed in mice and guinea pig preparations.
Calder et al. (1974) studied effector cells from human peripheral blood
and, although they also considered them to be neither T nor B lympho­
cytes, they did not observe glass or plastic adherence of the human
effector cells.
Rachelefsky et al. (1975) observed a correlation between
antibody-dependent lymphocyte cytotoxicity and the number of B cells in
normal and immunodeficient people, suggesting that the effector cell could
be a nonimmunoglobulin bearing B lymphocyte.
Until recently, no experimental evidence existed to indicate a role
for this cytotoxic mechanism in viral infections.
Allison (1972a) per­
formed histological studies on liver biopsies of human patients with
Australia antigen -pusi Live, ul iuically active hepatitis ar.d or. brains frc%
mice with virus encephalitis.
He observed that a high percentage of the
infiltrating cells morphologically resembled the effector cells of antibody-madiated lymphocyte-dependent cytotoxicity, and postulated that this
mechanism could play a role in virus-associated immunopathologic reac­
tions.
Ramshaw (1975) has now provided an animal model where antibody-
mediated lymphocyte cytotoxicity seems to be the mechanism for immune
cytolysis of infected cells; nonadherent spleen cells from herpes simplexinfected mice lyse ^icr-labeled virus-infected target cells.
This activi­
ty was not affected by treatment of the cells with anti-e serum but it was
28
abolished by anti-Ig serum.
The observation that normal spleen cells can
lyse antibody-coated target cells seems to indicate that the effector cell
is not an inrnune B cell but a nonspecific, nonlg-bearing lymphocyte.
Russe! et al. (1975) claimed that a similar mechanism is operative in
humans with recurrent cold sores due to herpes simplex virus infection.
They observed specific release of s^Cr from infected target cells after
incubation with either lymphocytes from people with herpetic infection or
a mixture of sera from patients (with high CF titers) and normal lympho­
cytes.
They did not establish the role of T cells in affected subjects,
however, and therefore could not rule out the possibility of more than
one operational mechanism.
The preceding reports emphasize the fact that many different cyto­
toxic mechanisms can be operable in different animal species or even in
different virus-host interactions within a single species.
(4) Cytotoxicity mediated by complement and normal lymphocytes —
Perlmann and his coworkers (reviewed by Perlmann and Holm, 1969) have
observed a cytotoxic effect by lymphoid cells iriyyercu L»y target cellbound complement.
He described two different mechanisms; one is a macro-
phage-mediâtèu destruction of C'3carrying target cells (related to phago­
cytosis, following a step in iirmune adherence) and the other is a small
lymphocyte-mediated destruction of C'7-carrying target cells.
In both
cases, the complement is bound to IgG coated target cells.
These mechanisms differ from complement-mediated cytolysis because a
longer period of time is required (20 hrs), the effector immunoglobulin is
an IgG, the reaction requires antibody at much smaller concentration
(1000-fold less) and alteration of the Fc fragment of this antibody does
29
not reduce the capacity to fix complement for this reaction.
The mecha­
nism has only been observed in vitro with chicken erythrocytes and its
biological significance is unknown.
(5) Macrophage cytotoxicity — Many of the phenomena produced by
cytotoxic lymphocytes can be mimicked by macrophages (Waksman, 1974).
Two
main mechanisms are operational; macrophages bearing cytophilic antibody
or macrophages activated by mediators released from stimulated lymphocytes
(or by other unknown mechanisms during infection).
Macrophages coated with cytophilic antibody can duplicate many of
the functions of sensitized T lymphocytes and, particularly, cytotoxicity.
Evans and Alexander (1970) reported that peritoneal macrophages from
lymphoma immune mice could inhibit the growth of these cells in vitro.
This reaction required cell contact, and it was specific.
Granger and
Weiser (1966) reported the presence of cytophilic antibody on these
macrophages ("armed" macrophages) and suggested that cytophilic antibody
was responsible for this specificity of action.
Even if cytophilic anti­
body is responsible for tne antigenic recogniLiun of tiicse specific
"killer" macrophages, there is evidence indicating that the macrophages
may require activation by an immunological reaction» mediated by specific
T
cells (Nelson, 1972; Cerottini and Brunner, 1974).
If such a "specific
macrophage-arming factor" (Cerottini and Brunner, 1974) actually exists,
the distinction between this type of macrophage cytotoxicity and that of
"activated" macrophages would not be so sharp.
In Evans and Alexander's experiments (1972) it was shown that after
the "armed" macrophages had been in contact with the specific target
cells, they acquired the capacity to lyse other susceptible cells in a
30
nonspecific way.
This suggested a two-step mechanism involved in the
activity of macrophages from immune animals; first, "armed" macrophages
interact with specific antigens and, second, this interaction leads to
their activation in a nonspecific way ("activated" macrophages).
This process shows great analogy with the concept of "activated"
macrophages put forward to account for the increased nonspecific bacteri­
cidal activity of macrophages from immune animals (Mackaness and Blanden,
1967; Mackaness, 1971).
This acquired resistance to a large variety of
microorganisms has been observed mainly in association with intracellular
bacterial infections such as Listeria monocytogenes and Brucella spp.
(reviewed by Nelson, 1969; Mackaness, 1971).
It can be induced by live
organisms only, is associated with delayed hypersensitivity, is to some
extent nonspecific, and can be transferred passively by T lymphocytes only
if the recipient has normal mononuclear phagocytic cells.
There are very few reports of acquired nonspecific resistance after
viral infections.
Blanden (1970, 1971a,b), working with ectromelia virus
infection in mice, showed that T cells were responsible for initietLitiy
recovery and observed that nonspecific radiosensitive monocytes inade a
major contribution to the effector mechanism (i.e., destruction of viral
foci).
Blanden and Mims (1973) reported macrophage activation that ivas
demonstrated by an increased ability to kill Listeria monocytogenes after
infection of mice by ectromelia or lynhocytic choriomeningitis virus.
Pathak et al. (1974) demonstrated similar mechanisms in chickens; perito­
neal macrophages from chickens recovering from fowlpox virus infection
showed Increased resistance to infection by Newcastle disease virus and
Salmonella gallinarum.
31
Alexander and Evans (1971) reported an intriguing observation;
endotoxin (or its hydrolysis product, lipid A) and viral double stranded
RNA have the capacity to render mouse peritoneal macrophages cytotoxic to
other mouse cells.
Their study indicated that "activation" could occur
under both in vivo and iin vitro circumstances.
Molecular mediators
Certain products of sensitized lymphoid
cells cultured in vitro with specific antigens can simulate some of the
phenomena associated with the expression of cell-mediated immunity.
These
products, different from immunoglobulins and producing their effects
independently of the continued presence of antigen, have been grouped
under the generic name of "lymphokines".
Table 1 is a list of these factors published in a recent report of a
World Health Organization scientific group (World Health Organization,
1973).
Detailed reviews of the literature concerned with these mediators
have been published by Yoshida and Cohen (1974), Greaves et al. (1973)
and Bloom and Glade (1971) among others.
These systems have not had ex­
tensive application as in vitro correlates of cell-mediated ifrsaunity to
viral infections.
Macrophage migration inhibition factor (MIF) has been used to evalu­
ate cell-mediated immunity to herpes simplex virus in humans (Simmons
et al., 1974; Wilton et al., 1972), fibroma virus in rabbits (Tompkins
et al., 1970), rubella virus in guinea pigs (Morag et al., 1974), and
influenza virus in humans and guinea pigs (Waldman et al., 1972).
The immunologically specific production of interferon by stimulated
lymphocytes was reported after herpes simplex infection in humans
32
Table 1.
Biological activities of products of activated lymphocytes
A. Affecting macrophages
Migration inhibition factor
(MIF)
inhibits the migration of
normal macrophages
Macrophage aggregation factor
(MAP)
agglutinates macrophages
in suspension
Macrophage chemotactic factor
(MCF)
causes macrophages to
migrate through micropore
filter along gradient
Macrophage resistance factor
(postulated)
renders macrophages nonspecifically resistant to
infection with certain
bacteria and viruses
cytophilic antibodies
confer on macrophages
specific reactivity with
antigen
B. Affecting lymphocytes
Blastogenic or mitogenic factor
(BP or MP)
induces blast cell trans­
formation and tritiated
thymidine incorporation
in normal lymphocytes
Potentiating factor
(PF)
augments or enhances on­
going transformation in
mixed lymphocyte culture
or antigen-stimulated
cultures
Cell cooperation or helper factor
produced by T cells, in­
creases the number or
rate of formation of Abproducing cells in vitro
Suppressor factor (postulated)
inhibits activation of,
and/or antibody produc­
tion by, B cells
^From: Cell-mediated Inr.unity and resistance to infection. World
Health Organization Technical Report Series No. 519. 1973. WHO, Geneva.
33
Table 1.
(Continued)
C. Affecting granulocytes
Inhibition factor
inhibits the migration of
human buffy coat cells or
peripheral blood leuco­
cytes from capillary
tubes or wells in agar
plates
Chemotactic factor
causes granulocytes to
migrate through micropore
filter along a gradient
D. Affecting cultured cells
Lymphotoxin
(LT)
cytotoxic for certain
cultured cells, eJg.,
mouse L cells or HeLa
cells
Proliferation inhibition factor
and cloning inhibition factor
(rIF,CLir)
inhibit proliferation of
cultured cells without
lysing them
protects cells against
virus infection
Interferon
E. Producing effects in vivo
Skin reactive factor (possibly
a combination of several of
the above activities)
Macrophage disappearance factor
(SRF)
in normal guinea pig skin
induces indurated skin
reactions that are histo­
logically similar to delayed-type hypersensitiv­
ity reactions
injected intraperitoneally, causes macro­
phages to adhere to
peritoneal wall
34
(Rasmussen et al., 1974; Val le et al., 1975) and vaccinia virus in humans
(Epstein et al., 1972).
This "immune" interferon is sensitive to pH 2 and
of higher molecular weight than the virus induced interferon (Falcoff,
1972; Sal vin et al., 1973).
As mentioned before, Kanra and Vesikari (1975) reported production of
a lymphotoxin-like activity in cultures of human lymphocytes stimulated by
rubella antigens.
Other jn^ vitro correlates of cell-mediated immunity
Ennis
(1973) and Simmons et al. (1974) have reported a plaque size reduction
technique as a measure of viral cell-mediated immunity.
Immune guinea
pig and mouse spleen cells prevented the spread of herpes simplex virus in
cell monolayers.
This technique correlated well with migration inhibition
factor and skin hypersensitivity (Simmons et al., 1974).
The reaction is
solely dependent on T cell function (Ennis, 1973).
A similar technique was reported by Rouse and Babiuk (1975) with
immune bovine lymphocytes and IBR virus infected cells.
Bovine T
lymphocytes were reported to be the effector cells (Rouse and Babiuk,
1974b).
Local iimunity
A very important part in resistance to infection
is played on the mucosal surfaces by the secretory immunologic system.
TgA is the predominant class of immunoglobulins in all secretions of man
and many mammalian species.
is due to two factors:
The relative increase in concentration of IgA
(1) local synthesis in submucosal lymphoid tis­
sues; and (2) secretion from plasma into mucosal surfaces with the aid of
a special polypeptide, the secretory component (Spiegelberg, 1974; Rossen
et al., 1971).
35
A secretory IgA system has also been identified in the bovine species
where IgA is the main secretory immunoglobulin in nasal and lacrimal se­
cretions, saliva and spermatic fluid (Mach and Pahud, 1971).
Its concen­
tration is very low in milk and colostrum, however, where IgG is the main
immunoglobulin class (Porter, 1973; Butler, 1973).
Although the concen­
tration of IgA is relatively high in intestinal secretions, IgM is usually
the predominant immunoglobulin in the bovine gastrointestinal tract
(Porter et al., 1972; Porter, 1973).
The secretory antibody system can function independently from the
central antibody system and can be readily activated by local application
of antigen (Rossen et al., 1971; Todd, 1973).
This fact is of extreme
importance in many viral infections where the primary site of virus
multiplication is either the intestinal or the respiratory tract.
Studies
of respiratory viral infection in man have shown that presence of antibody
in nasal secretions is the best correlation of protection against viruses
such as parainfluenza (Smith et al., 1966), rhinovirus (Rossen et al.,
lybb), and influenza virus ^riciriri el al., i5oo, Mâluwân êL al., 1909).
Similar observations have been reported for viral intestinal infections
such as poliovirus infection (Qgra et al., 1968).
Studies with parainfluenza-3 virus in cattle also seem to indicate
that secretory IgA is an important response to respiratory virus infection
and that local respiratory iirmunity after intranasal vaccination confers
better protection from infection than circulating antibody (Morein, 1970;
Gutekunst et al., 1969; Marshall and Frank, 1971; Frank and Marshall,
1971).
36
The induction of secretory or "local" antibody is not, however, the
only specific immune response of the mucosal surfaces to viral infection.
There are reports indicating that cell-mediated immune responses can also
occur locally in the respiratory tract after intranasal infection without,
or with a much smaller, generalized response.
Waldman et al. (1972) re­
ported the occurrence of local cell-mediated immune responses to influenza
virus in man and guinea pigs, and Morag et al. (1974) reported similar
findings after intranasal inoculation of guinea pigs with rubella virus.
As pointed out before, nonspecific mechanisms are also probably
important in local protection, not only with factors such as muco-ciliary
barriers, temperature or virus inhibitors, but also nonspecific effects of
infection such as cellular resistance, interferon production and phago­
cytosis (Merigan, 1974).
Recovery from virus infection
The nonspecific mechanisms of resistance described early in this
review determine, in many cases, the outcome of a viral infection.
How­
ever, after a viral infection is deft tri Lively established with active
virus multiplication occurring in a fully susceptible host, active defense
responses, both specific and nonspecific^ must interplay to counteract the
infection; fever, phagocytosis, inflammatory reactions and associated
reactions, are among these early responses.
Interferon production is one of the first body responses to most
viral infections.
Interferon is produced locally at the site of initial
infection, but may be distributed in the circulation to exert a protective
effect at sites distant from those where the virus is multiplying (Fenner
et al., 1974).
The production of interferon may be detected a few hours
37
after infection and days before the specific immune response is operation­
al.
Interferon may persist for several days.
High concentrations of
interferon are present in infected tissue at the time virus concentrations
begin to fall (Bellanti and Artenstein, 1964; Baron, 1970).
Passive
transfer of interferon indicated that circulating interferon can reduce
the viremia and reach target organs to protect cells against subsequent
seeding of virus from the blood (Baron et al., 1966a,b; Pinter, 1966).
In the absence of specific inhibitors of interferon production, or
genetically deficient experimental animals, it is very difficult to evalu­
ate the importance of interferon in promoting recovery.
Decreased func­
tion of the interferon system caused by altered body temperatures, stress,
chemical inhibitors and different virus strains has led to impaired re­
covery of laboratory animals (Baron, 1970).
Genetic differences in inter­
feron responsiveness determined the severity of arbovirus B infection in
mice (Hanson et al., 1969).
Experiments with immunosuppressive agents have shown that, in some
instances, normal levels of interferon fail Lu jjrùLect animals frcm viral
infections if the specific immunologic reactions are rendered inoperative
by immunosuppression (Glasgow, 1971).
The appearance of specific immunologic responses brings into play the
most powerful defense mechanisms to eliminate the infection, but it is
often difficult to discern which effector mechanisms are more important in
particular infections.
Experiments with immunosuppression and restoration
by antibody, as well as clinical observations in immunodeficiency states,
have revealed that viruses that promote systemic disease with viremia are
probably controlled primarily by circulating antibody.
Enterovirus and
38
arbovirus infections fall into this category and these viruses even fail
to elicit a cell-mediated irrenune response in most cases (Allison, 1972a,b;
Notkins, 1974).
On the other hand, viruses which spread from cell to cell
through intercellular bridges, and which usually elaborate specific anti­
gens on the cell surface, are more likely to be protected from circulating
antibody and to elicit cell-mediated immune responses (Allison, 1972a,b;
Notkins, 1974; World Health Organization, 1973).
Herpesviruses, pox­
viruses and some myxoviruses, as well as oncogenic viruses (which although
showing vertical transmission very often induce specific antigens on the
cell surface) would be included in this group (Notkins, 1974).
The ques­
tion arises concerning the mechanism of action of cell-mediated immunity.
How do cell-mediated immune responses act in vivo to eliminate infection?
What is the in vivo function of the in vitro correlates of cell-mediated
immunity?
The following is an excerpt from the World Health Organization
Scientific Group Report (World Kealth Organization, 1973):
..."sensitized lymphocytes circulate and become stimulated to
synthesize biologically active products when they interact with
specific antigen in the tissues, the chemotactic fauLor mlynt
be responsible for the accumulation of monocytes and macrophages
at sites of antigen implantation; the blastogenic factor might
activate adjacent lymphocytes in the tissues, i.e., act as an
amplification mechanism for the few sensitized cells at the
site; the migration inhibition factor might arrest macrophages
at the site of the reaction; and the postulated macrophage re­
sistance factor might account for the increased nonspecific
resistance to infection of macrophages, although for this effect
cell-cell interaction might be required. Infected or otherwise
antigenically altered cells might be destroyed or limited in
their growth by lymphotoxin and the proliferation inhibition
factors; and uninfected cells might be protected from viral
infection by interferon."
This is a qualitative interpretation of the events that may occur in
certain cell-mediated reactions based on the existence of the lymphokines.
39
However, the interpretation is purely speculative since it has not been
tested in vivo.
Salvin et al. (1973) reported the isolation of migration
inhibitory factor and "inmune" interferon from blood within hours after
intravenous inoculation of tuberculin into BCG-sensitized mice, but this
observation sheds little light on the mechanism of action in vivo.
Efforts to isolate lymphokines from tissues, on the other hand, have con­
sistently been unsuccessful (Yoshida and Cohen, 1974).
Cytotoxic reactions of lymphocytes are very likely to be important in
the control of virus infections (World Health Organization, 1973), partic­
ularly in the case of oncogenic or noncytolytic virus infections, but
their role in cytolytic infections is difficult to assess.
If lymphocyte-mediated cytotoxic reactions do take place, a coopera­
tive role of circulating antibody and complement in the destruction of
virus-infected cells cannot be ruled out.
Kibler and Meulen (1975) and
Russell et al. (1975) have reported production of cytotoxic antibody in
people after measles and herpes simplex infections.
A whole range of
virus-infected cells can be destroyed in vitro py antiviral antibuûy cmû
complement (Porter, 1971; Notkins, 1971).
Lodmall et al. (1973)» working
in an entirely in vitro system, showed that the only wsy to "cure" herpes
simplex-infected cell cultures was by a cooperative effect of leukocytes
and antibody plus complement; antibody and complement alone failed to
stop the spread of the virus from cell to cell, and leukocytes alone
halted the spread only temporarily.
40
Immunosuppression During Viral Infections
The depression of immune responses during viral infections has re­
ceived increasing attention in the last 10 or 15 years.
The first report
by Von Pirquet (1908) on the abrogation of tuberculin=delayed hyper­
sensitivity by measles infection was forgotten and the subject neglected
for more than half a century.
After Old et al. (1960) reported that
Friend leukemia virus impaired the ability of infected mice to produce
antibodies to sheep red blood cells, a number of investigations were ini­
tiated in different laboratories and conclusively demonstrated that viral
infection can interfere with normal itranune function.
Notkins et al. (1970), Sal aman (1970) and Dent (1972) have published
very extensive reviews of the work on immunosuppression caused by onco­
genic and nononcogenic viruses.
They included reports of decreased
humoral responses to a variety of nonreplieating antigens during infection
with a number of oncogenic viruses such as Gross virus, Moloney virus,
Frisnd virus, Rauscher virus and Marek's disease virus.
Lymphocytic
choriomeningitis virus, Newcastle disease virus, Junin virus, murine
cytomegalovirus and Aleutian mink disease virus were among the nononco­
genic viruses reported to produce depression of humoral responses.
Most of the early work dealt with antibody responses only, but in a
few cases where call-mediated immune responses were studied, host versus
graft reactions were shown to be impaired in infections with Gross leu­
kemia virus and Marek's disease virus.
Persistent skin hypersensitivity
reactions were temporarily abrogated during infections by measles.
41
influenza, chickenpox and poliovirus.
Impaired phagocytosis was reported
during ectromelia, lymphocytic choriomeningitis and Dengue virus infec­
tions.
The rather extensive list of immunosuppressive viruses comprises
agents with a wide range of biological effects, both in disease causing
ability and immunosuppressive potential.
This immediately suggests dif­
ferent mechanisms of action by various viruses, an idea supported by the
still inconclusive evidence concerning their effects on the iimune sys­
tem.
Several mechanisms have been proposed:
(1) Antigenic competition — The possibility that the immunosuppres­
sive virus competes as an antigen with the test antigen was raised during
the early work with leukemogenic viruses.
This hypothesis has been
largely discarded due to the poor antigenicity of most leukemia viruses,
differences in time intervals and effects on secondary responses from the
classical characteristics of antigenic competition, and the lack of cor­
relation between virus recovery and immunosuppression in some systems
(Dent, 1972).
(2) Competition for a cossîîofî stem cell -- This hypothesis was
postulated by Siege! and Morton in 1965 to explain the fact that most
leukemogenic viruses do not alter ongoing antibody synthesis and depress
primary responses to a greater degree than secondary responses.
They also
suggested that prolonged antigen and adjuvant treatment impaired leukemogenesis by previous differentiation of susceptible stem cells into anti­
body-forming cells (Siegel and Morton, 1967).
Bennet and Steeves (1970)
observed a differing susceptibility to cortisone treatment in precursors
of plaque-forming cells and Friend virus focus-forming cells suggesting
42
that, at least in the sheep erythrocyte-Friend virus system, antigenic
competition for a common precursor cell does not exist.
(3) Alteration of immunocompetent cells -- Although this simple ex­
planation probably does not apply to many of the long-latent-period leukemias (Dent, 1972), it agrees with the findings in many of the acute,
nonortcogenic virus infections.
Many of the viruses known to cause immuno­
suppression possess the ability to grow in leukocytes (Gresser and Lang,
1966).
Many of these infections usually result in metabolic depression of
the cells.
Will ems et al. (1969) observed reduction of PHA responses in
human leukocytes infected by poliovirus, echovirus, influenza virus,
Sendai virus, Newcastle disease virus, vesicular stomatitis virus, herpes
simplex virus, vaccinia virus, adenovirus and papilloma virus.
Vaccina­
tion of man with live rubella virus vaccines also results in temporary
inhibition of lymphocyte responses to PHA (McMorrow et al., 1974", Vesikari
and Buimovici-Klein, 1975).
Kantzler et al. (1974) observed temporary
loss of preexisting skin hypersensitivity to a variety of antigens and
suppression of the lymphocyte response to PHA drier influenza virus in
fection in man.
These studies have been considered as indicators of sup­
pression of T cell responses, although no investigations were made of the
possible influence of the infection on B cell functions.
However, Bro-
0(6rgensen et al. (1975) in a more comprehensive study involving several
parameters of T and B cell function in lymphocytic choriomeningitis virusinfected mice, demonstrated selective suppression of T-cell function.
Similar results were reported by McFarland (1974b)on studies of measles
infection in mice.
He reported decreased antibody response to carrier-
hapten conjugates due predominantly to a defect in T helper cells
43
(although B cells may have been affected to a lesser extent).
Fuccillo
et al. (1974) observed that children with congenital rubella infections
usually show antibody titers to this virus, but no cell-mediated immunity.
This finding may possibly explain the persistent virus excretion during
this condition.
Murine cytomegalovirus seems to produce more generalized effects on
the immune system.
Howard and Najarian (1974) and Howard et al. (1974)
reported depression of both humoral and cell-mediated immunity in mice.
The mechanism was associated with cytolytic infection of the lymphoreticular system which showed necrotic lesions and maximal virus content
at the time of maximal suppression of immune response.
The observation of low antibody responses to Newcastle disease vac­
cines in chickens with serological evidence of infection with infectious
bursal disease suggested the possibility of B cell deficiencies caused by
the virus.
This observation is plausible since the virus is known to
specifically affect the bursa of Fabricius (Harris, 1974).
(4) Alteration of macrophage function -- riotkins (1965) observed LliaL
a functional impairment of the reticuloendothelial system was the only
clinical pathological observation in mice infected with lactic dehydro­
genase virus (LDV).
LDV causes changes in immune responses of mice
usually evidenced as enhanced antibody synthesis, depressed cell-mediated
responses and depressed phagocytic function (Notkins et al., 1970).
These
observations are consistent with the finding that maximal concentrations
of LDV virus are usually associated with macrophages (Evans and Sal aman,
1955).
44
Mumps vaccine is another example of a live virus vaccine that can
affect preexisting delayed hypersensitivity.
Hall and Kantor (1972)
recently showed that this effect is caused by inhibition of the nonspe­
cific effector cells (monocytes) and not of the specifically sensitized
cells (T cells); picryl-albumina sensitivity was successfully transferred
from the infected guinea pig with depressed skin sensitivity to normal
recipients.
Transfer to infected animals of a new specificity, bovine
gammaglobulin, was not as successful as transfer into controls.
K.leinerman et al. (1974) observed that herpes simplex and influenza
virus infections in man greatly reduced the chemotaxis of blood monocytes.
This finding suggested that the action of these viruses on cell-mediated
inrnune responses could be due to their influence on mononuclear effector
cells.
Poliovirus has also been shown to replicate in human leukocytes and
to inhibit their response to PHA (Willems et al., 1959).
Soontiëns and
Van der Veen (1973) have presented some evidence indicating that poliovirus inhibits the response to PHA by suppressing the enhancing effect or
macrophages rather than by direct action on lymphocytes.
These reported experiments indicate that, at least In acute nononcogenic viral infection, immunosuppression could be due to direct attack on
cells of the lymphoid system, since most of these viruses have been shown
to multiply in vitro in leukocyte cultures.
The extent of the suppressive
response probably depends on the degree of functional impairment of the
lymphoreticular system.
Although most of the studies described above have
not been extended to both humoral and cell-mediated responses, the latter
45
seem to be more susceptible to the mild immunosuppressive effects clini­
cally observed during viral infection or after vaccination of man.
Bovine viral diarrhea (BVD) virus has an affinity for cells of the
immune system and destruction of lymphoid tissues can be observed in
natural cases of the disease (Ramsey, 1956; Pritchard, 1963).
There are a
few reports on field cases of the disease or on postvaccinal reactions
that suggest that immunosuppression can occur in cattle during BVD infec­
tion (Peter et al., 1967; McKercher et al., 1968; Brown and Andrews,
1973).
Shope (1964) and Muscoplatt et al. (1973a) contended that fatally
infected cattle failed to develop BVD neutralizing antibodies even after
protracted clinical illness.
This failure of an antibody response does
not always occur, however, as indicated by the author's experience as well
as the experience of others with field cases of the disease (Brown and
Andrews, 1973).
The best evidence for a possible immunosuppressive role of BVD infec­
tions came from studies of the ability of the virus to grow in leukocytes
and its role in lymphocyte responses to PHA.
Truitt ctnti Slieoniiicistsr
(1973) reported that BVD virus can multiply in vitro in bovine lymphocytes
and macrophages.
PHA stimulation of infected cells usually results in
increased virus titers.
Cells from both immune and susceptible cattle
supported virus growth equally well.
Muscoplatt et al. (1973a) reported
inhibition of lymphocyte response to PHA after in vitro or in vivo infec­
tion.
There are no reports, however, on the effect of BVD virus infection
on specific immunologic responses.
46
Immune Responses in Cattle to IBR and BVD Viruses
Recovery from natural infection with BVD ordinarily confers solid,
long-lived immunity to subsequent infections (Pritchard, 1963).
High
serum antibody titers usually result from either infection or vaccination
with live virus.
A number of serological techniques have been used to
quantitate those responses (reviewed by Classick, 1970).
Complement
fixing antibodies are detectable approximately 3 weeks after infection and
persist for only a few weeks or at most up to a few months.
Neutralizing
antibodies, first detected 2-3 weeks after infection, tend to persist for
much longer periods of time (Ruckerbauer et al., 1971).
In utero infec­
tion of bovine fetuses 168 days or older results in development of neu­
tralizing antibodies (Casaro et al., 1971).
This fetal response is com­
posed of both IgM and IgG immunoglobulins (Braun et al., 1973).
Rosenquist (1973) reported that most calves infected intravenously with
BVD virus failed to produce detectable circulating interferon.
There are no reports of secretary in^nunity or cell-mediated immune
responses to BVD virus.
The report by Truitt and Shechmeister (1973) that
leukocytes from "immune" cows supported viral growth at the same rate as
cells from normal cows, suggested that those lymphocytes lacked antiviral
properties.
The immunity elicited to the respiratory form of IBR virus infection
is strong and long lasting, but immunity to the genital form affords short
term protection (McKercher, 1973).
A carrier state follows active
infection and, despite strong immunity to reinfection, there is inter­
mittent shedding of virus for long periods of time (Snowdon, 1965; Bitsch,
47
1973).
Almost all of the early neutralizing antibody response to IBR
virus is composed of complement-dependent IgM, later followed by noncomplement-dependent IgG (Potgieter, 1975).
This early IgM antibody is al­
ready present at high titers at 8 days postinoculation.
Cell-mediated immune responses to IBR virus, as measured by lympho­
cyte transformation, are first detected at 5-6 days postinoculation,
peaking between 7 and 10 days postinoculation, and vanishing before the
third week postinoculation (Davies and Carmichael, 1973; Rouse and Babiuk,
1974a).
Nonadherent, nonimmunoglobulin bearing small lymphocytes, pre­
sumably T cells, are responsible for the antigen specific stimulation and
for inhibition of viral plaques in cell monolayers (Rouse and Babiuk,
1974b).
Interferon is produced in response to IBR infection or vaccination
with live virus vaccines.
Rosenquist (1973) reported production of high
titers of interferon in serum after intravenous inoculation of virulent
virus and lower titers after intramuscular or intratracheal inoculation.
Todd et ai. (ly/z) detected high titers uf itiLerTetOn in nasal secretions
of calves following intranasal vaccination with a vaccinal strain.
High
titers were present during the period between the third and the eighth
days postinoculation.
and the eighth days.
Low serum titers were observed between the fourth
48
MATERIALS AND METHODS
General Methods
Cell culture media and reagents
Minimum essential medium^ (MEM, Eagle) with Earle's salts
Earle's balanced salt solution^ (EBSS)
Hanks' balanced salt solution^ (HBSS)
Medium 199 with L-glutamine and 25 mM Hepes buffer^ (M 199)
Phosphate buffered saline pH 7,5 (PBS)
0.1 M KCl-HCl buffer pH 2 and pH 3
2
Fetal calf serum (PCS) - A single batch, screened for virus and
mycoplasma, and free of BVD and IBR antibodies, was used in virus isola­
tion, virus propagation and all the lymphocyte cultures.
3
Penicillin was used at a concentration of 100 units/ml.
Streptomycin" was used at a concentration of 100 vg /ml.
3
Fungizone was used at a concentration of 0.25 yg/ml.
Gentamicin* was used at a concentration of 50 vy/ml.
Bovine testicle cell cultures
Calf testicles were obtained from a commercial abattoir Ir: Dubuque,
Iowa.
The parenchyma of these organs was removed, minced and trypslnizsd
^Grand Island Biological Co., Grand Island, New York.
p
International Scientific Industries Inc., Cary, Illinois.
^E. R. Squibb and Sons s New York, New York.
^Schering Corp., Port Rsading, New Jersey.
49
in a 0.25% trypsin^ solution.
Dispersed cells were suspended at a con­
centration of 1.5 million cells/ml in a growth medium that consisted of
MEM plus 0.22% sodium bicarbonate, 10% fetal calf serum, penicillin and
streptomycin.
plates
2
The cells were dispensed into 60 mm plastic tissue culture
for virus isolation tests or into flasks
secondary cell cultures.
2
for later preparation of
For the preparation of secondary cell cultures,
monolayers were trypsinized with Trypsin-EDTA^ suspended at a 1 to 6
dilution in growth medium and dispensed into flasks for virus propagation
or into plastic tissue culture plates
interferon assays.
containing 24 (16 m) wells for
All cultures were incubated at 37 C in an humidified
CO2 cell culture incubator.
Each batch of bovine testicle cells was screened for the presence of
noncytopathogenic BVD virus by infecting 60 mm tissue culture plates with
a known number of plaque forming units (PFU) of BVD virus (NADL strain).
The plates were incubated for 5 days and observed for evidence of inter­
ference (reduction in number of PFU).
Viruses
Bovine viral diarrhea (SVD) viruses
were used.
Two strains of BVD viruses
The NADL Strain (number of tissue culture passages unknown)
was used for virus neutralization tests and for antigen preparation.
Strain 1015-74 was used for animal inoculation and antigen preparation.
^Difco Laboratories, Detroit, Michigan.
2
Falcon Plastics, Los Angeles» California.
3
Linbro Chemical Co., Inc., Msw Haven, Connecticut.
50
This strain was isolated at the Iowa Veterinary Diagnostic Laboratory from
a typical case of bovine virus diarrhea-mucosal disease where it affected
50% of a group of yearling calves with very high mortality.
Several
calves were necropsied in this outbreak with all of them presenting typi­
cal lesions of the disease.
BVD virus was repeatedly isolated.
viral or bacterial pathogen was recovered.
No other
This isolate was plaque puri­
fied and used at the third bovine testicle passage as a challenge strain.
Infectious bovine rhinotracheitis (IBR) virus
Strain VOL 353
A-72, isolated at the Iowa Veterinary Diagnostic Laboratory from a typical
case of infectious bovine rhinotracheitis, was used at the fifth bovine
testicle passage for animal inoculation and antigen preparation.
A type strain of IBR virus, originally obtained from the National
Animal Disease Center, Ames, Iowa, was used for antigen preparation and
serological tests.
Vesicular stomatitis virus (VSV)
VSV (New Jersey) was obtained
from Dr. L. Potgieter, Department of Veterinary Microbiology, Iowa State
University.
It was propagated in secondary bovine tesLiule lkHs aim used
for the assay of interferon.
Equine rhinopncumonitis virus (ERV)
A live modified vaccine
strain^ (Rhinomune) of ERV was used as a control antigen preparation in
the cytotoxic assay.
Virus propagation
Confluent secondary testicle cell monolayers were infected with IBR,
VSV or BVD viruses (approximately 0,1 to 0.5 PFU/cell).
^Norden Laboratories, Lincoln, Nebraska.
After allowing
51
viral absorption to proceed for 90 minutes at 37 C, maintenance medium
(MEM with 1% PCS) was added.
The cells were incubated until 90% were de­
tached, usually 24 hrs for VSV, 48 hrs for IBR and 5-6 days for BVD.
Cellular debris was removed by centrifugation at 1000 x g for 10
minutes at 4 C.
The challenge viruses and VSV virus were divided into
aliquots and held at -70 C until use.
The virus for antigen production
was held at 4 C until use.
Viral antigens for lymphocyte stimulation
IBR and ERV virus suspensions were centrifuged (50,000 x g; 4 C) for
1 hr; BVD virus suspensions were centrifuged (85,000 x g; 4 C) for 2 hrs.
An IEC B-60 centrifuge^ with an A-321 rotor was used.
The IBR and ERV
pellets were resuspended in 1/10 of the original volume; BVD pellets were
reconstituted at 1/15 the original volume.
The titers of the resuspended
virus were 10® PFU/ml for IBR and 5x10® PFU/ml for BVD.
titrated.
ERV was not
The IBR and ERV viruses were inactivated by heating at 56 C for
1 hr; BVD was inactivated by heating at 56 C for 2 hrs.
The antigen
preparations were held at -70 C until use.
Serum virus neutralization test
Antibody titrations were performed in a virus neutralization microtest, according to Jenney and Wessman (1973).
The only modification to
this method was the addition of complement; fresh guinea pig serum (C)
or heat inactivated guinea pig serum (HC) was added to the serum-virus
mixtures.
Briefly, two sets of 0.05 ml volume twofold serial dilutions of
the sera in EBSS were made in flat bottom tissue culture microtiter
1
'International Equipment Co., Needham Heights, Massachusetts.
52
plates J
IBR or BVD viruses (25 TCID50) were added in 0.05 ml volumes.
To one set of diluted serums, 0.025 ml of HC was added; 0.025 ml of C
was added to each well of the second set.
1 hr at room temperature.
The plates were incubated for
Bovine testicle cells in a 75 cm^ flask were
trypsinized during the neutralization period, suspended in 10-15 ml MEM
with 10% PCS and 0.05 ml of the cell suspension dispensed to each well of
p
the test plate. Two drops of mineral oil were added to each well, and
the plates incubated until 80-90% cell destruction was observed in the
control wells at which time the test was read.
Serum, cell, virus and C
controls were included.
Virus isolation
Virus isolations were conducted in primary bovine testicle cell
cultures in 60 mm plastic plates with an agar overlay.
The agar overlay
consisted of MEM with 1% Noble agar^ and 2% PCS plus antibiotics.
Cul­
tures were incubated for 8 days, and observed daily for appearance of
viral plaques.
Viral plaques, when observed? were harvested and the
virus seeded in Leighton tubes tor identificaxion.
virus ideniifitaLturi
was conducted by a direct fluorescent antibody method using BVD and !BR.
specific conjugates.^
^Falcon Plastics, Los Angeles* California.
^E. R. Squibb and Sons, New York, New York.
3
Difco Laboratories, Detroit, Michigan.
diagnostic Virology Section, Veterinary Services Diagnostic Labora­
tory, APHIS, Ames, Iowa.
53
When no viral plaques were observed, the cells were harvested and
repassed in primary bovine testicle cell cultures.
Samples were con­
sidered negative when no viral plaques were observed in this second
passage.
Interferon assay
Serum samples and supernatant fluids from lymphocyte cultures were
assayed for interferon activity by a plaque reduction method using VSV
as the indicator virus.
Twofold serial dilutions of the samples were
made in MEM with 1% fetal calf serum.
The dilutions, in 0.5 ml volumes,
were inoculated into plastic tissue culture plates^ possessing 24 (16 mm)
wells containing monolayers of secondary bovine testicle cells.
The cells were incubated for 18-20 hrs, the supernatant fluids were
aspirated and the cells washed twice with 1 ml of HBSS.
A suspension of
VSV in EBSS was adjusted to contain 200-300 PFU/ml and 0.1 ml was inocu­
lated into each well.
After incubation for 1 hr at 37 C, excess fluid was
aspirated and the cells were covered with 0.5 ml of agar overlay contain­
ing 1% PCS.
The plates were incubated at 37 C for 48 hrs, observed for
cytopathic effect and interferon titers were determined.
Experimental Procedures
Experimental animals
Eighteen 700-750 lb steers were used in the experiments.
These
steers were selected on the basis of complete absence of BVD and IBR
antibodies in a representative number of serum samples obtained from
1
Linbro Chemical Co., New Haven, Connecticut.
54
animals in the herd of origin.
Three of the steers were used in pre­
liminary experiments to determine susceptibility to BVD virus and to
select a challenge strain.
The remaining 15 steers were inoculated with
BVD virus alone, IBR virus alone or a combination of both agents (Table
2).
Table 2.
Design of the experiments.
BVD-l®
Experiment
Treatment of animals
ByD-2b
IBR
1
33Cf-331-336-346
2
335-337-BR
330-336
3
332-344
331-346
4
HRT-343
5
333-347
333-347-330-331
6
341-NT
341-NT-335-337
-
•
-
^First inoculation.
'^Reinoculation.
^Animal identification.
The BVD virus inoculum consisted of 10 ml of a 5 x 10^ PFU/ml viral
suspension in EBSS with 2% PCS administered intravenously.
The IBR virus inoculum consisted of 10 ml of a 5 x 10? PFU/ml viral
suspension in EBSS with 2% FCS administered by the intranasal routs.
IBR
virus was administered on the second day following BVD inoculation.
Body temperature and total white blood cell counts (WBCC) were de­
termined daily.
Nasal swabs and blood samples (with and without EDTA
55
anticoagulant) were collected for virus isolation.
The samples were col­
lected daily during the first week following inoculation and at variable
intervals thereafter=
Serum samples were collected daily during the first
week postinoculation for interferon assay.
Isolation of lymphocytes
Blood (200-250 ml) was collected by venipuncture into 1000 ml
Erlenmeyer flasks containing nine 6-inch wooden applicator sticks, at­
tached to each other near one end so as to fonn a vertical "cone" (Kay and
Kaeberle, 1972).
The blood was defibrinated by swirling by hand or by
shaking in a mechanical shaker for 10 minutes.
The defibrinated blood was transferred to 50 ml siliconized screw-cap
centrifuge tubes (25 x 150 mm) and centrifuged at 750 x g for 40 minutes
in a swinging bucket-type centrifuge.^
The buffy coat cells were aspi­
rated with a pipette and suspended in 2 volumes of sterile physiological
saline solution (0.85% NaCl).
An 8 ml quantity of this suspension was carefully layered onto 4 ml
9
of Ficoll-Hypaque (specific gravity 1.060) in à 13 x 100 ml plastic tiibc"
and centrifuged at 400 x g for 30 minutes.
The Ficoll-Hypaque solution was prepared by mixing 29,4 ml of Hypaque
solution (20 ml 50% Hypaque^ with 9.4 ml distilled water) with 53.33 ml of
a 10% Ficoll^ solution.
1
International Equipment Co., Needham Heights, Massachusetts.
^Falcon Plastics, Los Angeles, California.
Winthrop Laboratories, New York, New York.
^Pharmacia Fine ChemicalS; Piscataway, New Jersey.
56
The specific gravity was measured with a hydrometer and adjusted to
1.080 with distilled water.
The mixture was sterilized by filtration
through a 0.45 p membrane filter.^
The lymphocytes were aspirated from the interface with a pipette,
washed once in HBSS without Ca or Mg and suspended in the growth medium.
Further purification of lymphocytes
Lymphocytes for some of the cytotoxic assays were further purified
by filtration through cotton fiber columns (Kay and Kaeberle, 1972) or
nylon wool columns (Julius et al., 1973).
The cotton fiber columns were made by partially filling 12 ml disposable plastic syringes
2
with cheesecloth threads.
The columns were
washed with HBSS, and sterilized by autoclaving.
The nylon wool was removed from Leuko-Pak filters
3
and washed by
soaking the content of one filter in a 1 liter beaker containing glass
distilled water at 37 C.
Several changes were made over a period of 5
days. After manually expressing water, the wool was dried at 37 C.
U.b gr of dry wool ms packed into a 12 ml syrinye.^
About
Trie CuluiiinS were
sterilized by autoclaving, rinsed with sterile phosphate buffered saline.
pM 7.2, containing 5% fetal calf serum, and placed in an incubator at 37 C
for 1 hour before loading with cells.
Both types of columns were loaded with approximately 10® lymphocytes
in a 1 ml volume, washed into the column with 1 ml additional medium, and
^Millipore Filter Corp., Bedford, Massachusetts.
2
Monoject, Sherwood Medical Inds.s Deland, Florida.
3
Fenwal Laboratories, Morton Grove, Illinois.
57
incubated at 37 C for 45 minutes.
The columns were then washed slowly
with 20-25 ml of growth medium, the cells pelleted by centrifugation at
200 X g for 10 minutes and resuspended in fresh medium.
Plastic adherent and nonadherent cells were separated by incubating
5 to 8 X 10"^ Ficoll-Hypaque purified cells in 25 cm^ plastic tissue cul­
ture flasks for 1-2 hours.
Adherent cells were detached by trypsinization
with 0.25% trypsin for 20-30 minutes, washed in HBSS and resuspended in
M199.
Lymphocyte cultures
The growth medium for lymphocyte cultures consisted of M199 supple­
mented with 20% PCS, gentamicin and fungizone.
Ficoll-Hypaque purified
lymphocytes were suspended in growth medium to a concentration of 1.5 x
10® cells/ml, and 2 ml of this cell suspension were dispensed into dry
heat-sterilized glass culture tubes^ (13 x 125 mm) with Morton stainless
steel closures.
to the tubes.
Mitogens and antigens were then added in 0.1 ml volumes
O
Phytohemagglutinin-P (PHA-P) was used at 1:50 dilution and
poKeweea mitogen" (PWri) at a 1:10 dilution in growth medium.
Two sets of triplicate cultures were prepared for each of the anti­
gens, mitogens and control cultures.
Lymphocyte cultures were incubated
at a 30' angle in a 5% COg humidified atmosphere at 37 C.
One set of
cultures was used to measure DMA incorporation by stimulated lymphocytes.
The supernatants of the second set of cultures, incubated for the same
^ RTU #7824; Beckton; Dickinson and Co., Rutherford, New Jersey.
P
Difco Laboratories, Detroit, Michigan.
Grand Island Biological Co., Grand Island. New York.
58
period of time, were used for interferon assays.
The pooled supernatants
of each triplicate culture were kept frozen at -20C until assayed.
Termination of lymphocyte cultures for DNA analysis
One yCi of tritiated thymidine with a specific activity of 6.7 Ci/
in a volume of 0.1 ml, was added to each tube 22-24 hrs before
termination of the cultures.
Mitogen stimulated lymphocytes were incu­
bated for a total of 72 hrs.
Antigen stimulated lymphocytes were incu­
bated for 5 total time of 120 hrs.
At the termination of the incubation time, the cultures were centrifuged at 1000 x g for 15 minutes, the supernatant fluid decanted into a
radioactive waste container, and the cells resuspended in PBS.
After a
second centrifugation the supernatant fluids were decanted and the tubes
containing the cell pellets were immediately frozen at -20C and kept
frozen until prepared for DNA analysis.
DNA analysis
The cell pellets were thawed and 1 drop of newborn calf serum was
added to esch tube.
The cultures were Incn precipitated with cold 5%
trichloroacetic acid (TCA); 3 ml were added to each tube and maintained at
4 C for at least 1 hour.
The precipitates were sedimented by centrifuga­
tion at 1,500 X 9 for 20 minutes at 4 C, the supernatant fluid was dis­
carded and a 3 ml volume of TCA was added for a second precipitation step.
After centrifugation, the pellets were resuspended in 1 ml ice-cold
methanol, immediately centrifuged, the methanol discarded, and the re­
sidual precipitate dried at room temperature.
The precipitated DNA was
^New England Nuclear Corp., Boston, Massachusetts.
59
solubilized in 0.5 ml Soluene-100\ 10 ml of toluene-base scintillation
fluid (Permafluor^) was added, and the samples transferred into counting
p
vials.
Samples were counted for 10 minutes in a Packard TriCarb model
3375 liquid scintillation counter.^
Cytotoxic assay
Lymphocyte suspensions were assayed for cytotoxic activity by using
virus-coated chicken erythrocytes prepared according to a modification of
the technique described by Schirrmacher and Golstein (1973).
Fresh
chicken red blood cells (CRBC) were coated with viral antigens by means of
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (ECDI)^; 0.1 ml of a
50% suspension of washed CRBC was added to 1 ml of each viral antigen,
0.2 ml of ECDI (50 mg/ml in PBS) was added, and the mixture incubated for
1 hour at room temperature.
The cells were washed three times in normal
saline solution, and resuspended to approximately a 1% suspension (ad­
justed to a concentration such that after lysis of an aliquot in 39
volumes of distilled water, the mixture exhibited a 77-78% transmittance
in a Spectronic-2ù* spectrophuLunieLtt} in sal inc.
The cells ware labslsd
with sicr^ by incubating 1 ml of the CRBC suspension with 100 yCi of sicr
for 1 hour at room temperature.
The cells v.-ere washed twice in normal
saline solution and resuspended in 2 ml of normal saline solution.
^Packard Instrument Co., Downers Grove, Illinois.
p
Wheaton Scientific, Milville, New Jersey.
3
Sigma Chemical Co., St. Louis, Missouri.
^Bausch and Lomb Inc.* Rochester, New York.
5
ICN Isotope & Nuclear Div., Irvine, California.
60
Cytotoxicity was assayed by adding 1.5 to 2 x 10^ lymphocytes to 0.1
ml aliquots of labeled CRBC in 13 x ICQ mm sterile disposable plastic
tubes.
The final volume was adjusted to 0.8 ml.
The cells were centri-
fuged at 1200 x g for 1 minute to assure cell contact, and then incubated
for 18-20 hrs at 37 C,
At the end of the incubation period, the tubes
were centrifuged at 1500 x g for 10 minutes and the supernatant fluids
separated from the pellets.
Radioactivity of both cell pellets and super-
natants was determined in a Picker Nuclear Autowell II gamma counter.^
B cell counts
The percentage of immunoglobulin bearing cells in Ficoll-Hypaque
isolated lymphocytes was determined by staining the lymphocyte suspensions
with a rabbit antibovine globulin fluorescent conjugate.
2
Lymphocytes
were suspended in HBSS at a concentration of 10?/ml; 0.1 ml of the cell
suspension was added to a 0.2 ml volume of a 1:20 dilution of the con­
jugate and incubated at room temperature for 20-30 minutes.
The cells
were immediately washed in 5 to 7 ml of HBSS containing 0.1% sodium azide,
and resuspended in 3-4 drops uf HBSS.
The percar.tagG cf i%%unoglobulin-
bearing cells was determined by counting fluorescent cells and total cells
in an Ortholux II Leitz microscope with epi^'1uc>''=scGnt illumination.
Blood cultures
Bacterial examination of peripheral blood was conducted by inocula­
tion of venous blood into medium in commercial blood culture bottles.
Vicker Corp., New Haven, Connecticut.
p
Colorado Serum Co.s Denver, Colorado.
61
Bacto brain heart infusion blood culture bottles^ were used in experiments
I through IV.
Vacutainer Columbia broth blood culture bottles
2
and Vacu-
tainer Columbia broth with sodium polyanethol sulfonate (SPS) blood culture bottles
2
were used in experiment VI and in blood cultures from 10 of
the calves conducted twice after the end of the last experiment.
The skin was prepared by clipping the hair and disinfecting with 70%
alcohol or 1% tincture of iodine.
Blood cultures were directly performed
by jugular venipuncture with blood collection sets^ or by collecting the
blood in sterile disposable 30 ml syringes and subsequent inoculation of
the culture bottles.
Approximately 5 ml of blood were added to 50 ml of
medium.
The bottles were incubated overnight at 37 C and a loopfull of medium
2
was then plated onto trypticase soy agar with 5% citrated bovine blood,
1
MacConkey agar
and tergitol 7 agar.
2
Blood agar plates were incubated
both aerobically and anaerobically in a gaspack anaerobic jar.
2
agar and tergitol 7 agar plates were incubated aerobically only.
MacConkey
All
culture plates were incubated at 37 C for 72 hrs or until growth was ob­
served.
^Difco Laboratories. Detroit, Michigan,
p
Bio Quest, Cockeysville, Maryland.
62
RESULTS
BVD Virus Infection
Clinical observations
Infection of experimental cattle with BVD virus did not produce
significant clinical disease.
Some individual animals showed depression
and inappetence during the first 2 to 3 days and, occasionally, a mild
mucous nasal discharge was observed during the same period.
No signifi­
cant temperature changes were recorded other than transient elevation to
103-104 F in three of the calves on the third or fourth days following
inoculation.
Hematological changes
A leukopenic response was observed in all animals following infec­
tion.
Low WBCC were present on the first or second day postinoculation,
and reached the lowest counts on the fourth or fifth day.
Figure 1 shows
the mean WBCC for all calves inoculated with BVD virus in experiments 1
through 4.
The results are expressed as percenlayes uf Llic piciiioculaticn
mean WBCC (100%).
An increase in the number of immunoglobulin-bearing
lymphocytes was observed on the second day (Figure 2).
Virus isolation
Recovery of BVD virus from the blood of BVD inoculated calves was
inconsistent.
Virus was recovered on only five occasions, from blood
clots taken at 2, 3 or 4 days postinoculation.
(2-10 PFU/ml) in all cases.
Viral titers were low
Figure 1.
Mean responses to phytohemagglutinin-P of peripheral blood lymphocytes during the period
following inoculation of cuttle with BVD virus. The stimulation was measured by the
Incorporation of
thymidine. The results are expressed as stimulation ratios (cpm of
PHA stimulated lymphocytes/cpm of unstimulated lymphocytes) in calves infected with BVD
virus (PHA) and in control calves PHA (C). White blood cell counts (WBCC) are ex­
pressed as percentage deviation from the preinoculation levels (100%). The vertical
bars and figures in the upper left hand corner represent the percentages of animals
with positive bacterial blood cultures following BVD virus inoculation.
WHITE BLOOD CELL COUNT (% OF PRE-INOC.)
' r o O J 4 »
o
o
o
in
in
z
S
o
o
o
w
80
PHA STIMULATION RATIO (SR)
179
o
65
NUMBER OF CALVES
10
2
10
6
6
2
6
6
1
2
3
4
5
6
7
160
tz
UJ
CO 130
LU
a:
o
3
jU
120
u.
o
oa
LU
CO
Z
0
1 DAYS FOLLOWING BVD INOCULATION
Figure 2.
Percentage of immunoglobulin bearing cells in Ficoll-Hypaque
isolated lymphocytes in the period following BVD virus inocu­
lation. Results are expressed as percentage deviation from
the preinoculation values (100%). They are the mean values of
2, 6 or 10 animals as shown in the top portion of the figure.
66
Response of lymphocytes in culture to mitogens following BVD virus
infection
The response of lymphocytes to PHA and PWR was greatly reduced fol­
lowing infection.
Decreased responses were already apparent the first day
following inoculation, and lower responses were observed during the second
and third days.
Figures 1 and 3 show mean stimulation ratios (cpm stimu­
lated cells/cpm unstimulated cells) of peripheral blood lymphocytes of
calves inoculated with BVD virus.
It should be noted that although the
lowest mean stimulation ratios were observed on the third day postinoculation, the lowest cpm recorded corresponded to the second day.
The stimu­
lation ratios were lower on the third day due to increased label uptake
by unstimulated lymphocytes on that day.
The production of interferon by PHA stimulated lymphocytes was also
reduced by infection; the mean interferon titer in cultures of lymphocytes
(mean of five calves) were 128 on inoculation day and 16 on day 3,
PHA
stimulated interferon was susceptible to pH 2 but showed appreciable re­
sistance to treatment at pH 3 (Table 3).
Observation of bacteremia following BVD virus inoculation
During some of the preliminary BVD challenge experiments and during
experiment 1, almost all lymphocyte cultures started on the first few days
following BVD virus inoculation showed extensive bacterial contamination.
This repeated observation, and the failure to identify any cell culture
medium component or the reagents employed in lymphocyte separation as
possible sources of the contamination, led to consideration of the possi­
bility of bacteremia following BVD infection.
Therefore, blood cultures
in commercial blood culture medium were used to investigate this
67
IX
in
<A
o
a:
=3
to
DAYS FOLLOWING BVD INOCULATION
Figure 3.
Mean stimulation ratios of peripheral blood lymphocytes during
the period following inoculation of cattle with BVD virus. The
lymphocytes were treated with phytohemagglutinin-P (PHA), pokeweed mitogen (PWM) or left untreated in culture with stimula­
tion measured by the incorporation of ^M-thymidine.
68
Table 3. ' Reduction of interferon activity of samples dialyzed against low
pH buffer for 24 hrs. Results are expressed as range of the
percent reductions of the original interferon activity when both
untreated and treated samples were assayed at the same time
Supernatant fluid of
stimulated lymphocyte cultures
pH 3
pH 2
PHA
50-75
100
I BR
50-75
100
Serum
25-50
50-75
possibility on the third day following BVD virus inoculation in experiment
1 and in most of the other experiments thereafter.
The results of these
cultures are shown in Table 4 and Figure 1.
Control calves were included in all the experiments starting in ex­
periment 2.
They consisted of unino ulated calves or BVD immune calves
reinoculated with BVD or inoculated with IBR virus.
The contaminants isolated from lympnocyie cultures in experimeriL 1
consisted of Pseudomonas spp. that were resistant to all antibiotics
assayed with the sole exception of gentamicin.
Bacterial isolates from blood cultures yielded Bacillus spp. in all
cases, either in pure culture or associated with alpha hemolytic
Streptococcus spp. in two cases.
All the isolates were sensitive to
gentamicin and of variable sensitivity to other drugs including penicillin
and streptomycin.
The routine inclusion of gentamicin in the lymphocyte
cultures starting with experiment 2 prevented further problems with cul­
ture contamination.
69
Table 4.
Number of animals with positive bacterial blood cultures in the
period following BVD virus infection
Days following inoculation
Experiment
1
Calves
12
BVD
4/4*
Control
2
3
4
6
TOTALS
4/4*
3
4/4
4
5
6
-
-
_b
-
-
-
-
-
-
BVD
0/3
1/3
1/3
0/3
0/3
-
Control^
0/3
0/3
0/3
0/3
0/3
-
BVD
-
2/2
-
2/2
1/2
0/2
Control^
-
0/ 2
-
0/2
0/2
0/2
BVD
-
2/2
-
2/2
-
-
Control®
-
0/3
-
0/2
-
-
BVD^
2/2
2/2
-
1/2
-
0/2
Control^
0/2
0/2
-
1/2^
-
1/2^
BVD
5/9
11/13
5/7
5/9
1/5
0/4
Control
0/5
0/10
0/3
i/gh
0/5
1/4^
^Lymphocyte culture contamination.
^Not done.
^Two of the controls were calves reinoculated with BVD virus.
third calf was an uninoculated control.
'^Calves reinoculated with 5VD virus.
^Uninoculated control calves.
^Susceptible calves inoculated with BVD and IBR viruses.
^BVD immune calves inoculated with IBR virus alone.
"^Positive in medium with SPS only (calf #337).
The
70
In addition to the control calves included in each experiment, blood
cultures were made from ten of the calves 18 days and again 60 days after
the beginning of the last experiment.
Table 5.
Results are shown in Table 5.
Number of animals with positive bacterial blood cultures after
complete recovery from BVD virus infection
•
Days following inoculation^
18
60
Columbia broth
0/10
0/10
Columbia broth with SPS
5/10
2/10
Medium
It refers to the date of the start of the last experiment. The four
calves used in experiment 6 are included here plus six additional calves
used in prior experiments.
Nine isolates out of a total of 34 Bacillus spp. were identified as
Bacillus cereus.
Three additional strains were tentatively identified as
B^ subtil is, two as ^ brevis, one as ^ badius, and four as B^ coagulans
(Gibson and Gordon, 1974; Cowan eiiiû SLecl, 1S6G).
The ram&indcr were net
identified, and even the total number of species involved is uncertain
because of the variation in colony morphology^
It was estimated that at
least eight different species were isolated.
Production of circulating interferon
Interferon was detected in the serum of all BVD virus-infected cattle
during the first 5 days following inoculation.
on day 4 (Figures 4 and 5).
Peak titers were observed
Dialysis against pH 2 buffer for 24 hours did
not destroy ths interferon activity (Table 3),
Circulating interferon was
Figure 4.
Mean serum interferon titers (reciprocal of highest dilu­
tion of serum causing 50% or more reduction in the number
of VSV plaques) in the period following inoculation of
cattle with I BR or BVD virus.
Figure 5.
Production of interferon in cultures of lymphocytes stimu­
lated with IBR antigen. Results are expressed as mean
titers of interferon produced by lymphocytes from cattle
inoculated with IBR virus alone (IBR) or with ÎBR virus
following BVD virus challenge (BVD-IBR), .
72
Cà
3
4
5
6
7
10
8
DAYS FOLLOWING INOCULATION
64
i
32
IBR
/
lé
/
^
8
f
/ BVD-IBR
4
2
5
10
15
DAYS FOLLOWING IBR INOCULATION
20
73
not detected in any of the calves reinfected with BVD virus in experiments
2 and 3.
Specific immune response
Figure 6 shows the antibody response to BVD virus.
Both complement-
requiring and complement-independent neutralizing antibodies were first
detected 10 days following challenge and peaked at 24 days.
The addition
of complement resulted in maximum enhancement of neutralization at day 17,
when the mean complement-requiring antibody titer was more than twofold
higher than the complement-independent titer.
was not observed after 31 days.
Enhancement by complement
Reinoculation of cattle with BVD virus
resulted in a twofold increase in antibody titers in most cases, but the
addition of complement did not enhance this neutralizing activity.
Attempts to stimulate lymphocytes with BVD antigen in vitro were
unsuccessful.
Incubation of lymphocytes collected at different times
following infection, with varying quantities of BVD antigens (strains
1015 or NADL), did not result in an increase of
thymidine incorporation
or in immune formation of interferon.
Development of cytotoxic activity by peripheral blood leukocytes after BVD
infection
Lymphocytes from calves recovering from BVD infection exhibited cyto­
toxicity for BVD-coated CRBC as measured by s^Cr release.
Figure 7 shows
the development of this cytotoxic activity expressed as percentage of sicr
release (percentage of sicr release from CRBC in the presence of lympho­
cytes minus percentage of spontaneous release, i.e., that of CRBC alone).
The results for experiment 1 were reported separately from the other
experiments because the process of CRBC-coating was slightly changed in
74
1024r
512
256
128
64
32
16
8
4
2
7^
10
î
Figure 6.
20
30
60
DAYS FOLLOWING BVD INOCULATION
Development of complement-dependent (C) and complement-inde­
pendent (HC) BVD neutralizing antibody in serum of cattle
following inoculation with BVD virus. Results are expressed
as mean antibody titers (reciprocal of the highest serum dilu­
tion neutralizing 80% or more of the viral cytopathic effect)
in a micro-neutralization test.
40 -
EXPS.
II-IV
10^
4
6
H
10
12
14
16
18
20
22
24
DAY:S FOLLOWING BVD INOCULATION
Figure 7.
Development of cytotoxic activity in peripheral blood leukocytes of cattle following
inoculation with BVD virus. Results are expressed cis mean percentages of siQr release
± standard error from BVD-coated chicken erythrocytes.
76
the latter to reduce the amount of spontaneous release of s^Cr.
The
change, an increase in the amount of antigen, and a decrease in the amount
of ECDI, apparently decreased the sensitivity of the test.
However, the
modification increased the stability of the cells and reduced the spon­
taneous release of sicr from a variable 15-35% in experiment 1 to a much
more constant 9-14% in the remainder of the experiments.
In all experiments, the cytotoxic activity was first observed around
the seventh day following inoculation and peaked at 10 days.
decreased thereafter, vanishing 1 to 3 weeks later (Figure 7).
The activity
This
activity, however, was not specific; the percentage of siQr release by IBR
or ERV virus-coated CRBC was essentially the same as that of BVD coated
cells.
The nonspecificity of the reaction prompted an attempt to identify
the effector cells.
Passage of the Ficoll-Hypaque separated lymphocytes
throuth cotton or nylon wool columns resulted in either complete removal
or marked reduction oT the cytotoxic activity.
The resulting cell sus­
pensions were composed almost entirely of small lymphocytes with essen­
tially no iiiacrophaye- like cells aiiu a reduction in the nun,bar of immur.c
globulin-bearing cells.
Fractionation of the cells into plastic adherent and nonadherent
populations resulted in limited activity associated with the latter, but
marked activity associated with many of the adherent cell populations.
This increase was not constant, however, mainly due to clumping during
washing of some of the adherent cell preparations.
Table 6 shows mean activity of the different cell populations from
five different calves at 7, 11 and 14 days postinoculation.
77
Table 6.
Cytotoxic activity of peripheral blood leukocytes from calves
inoculated with BVD virus. Identification of effector cells
Cell populations
Days post-
Ficoll-
inoculation
Hypaque
1.90*
7
Nylon
Nonad­
Cotton
Wool
herent
Adherent
0.00
0.30
0.86
5.93
11
11.38
2.95
1.86
2.55
3.93
14
6.94
2.03
0.73
3.10
8.83
Percentage of sicr release {% siCr release in the presence of leuko­
cytes - % spontaneous sicr release by CRBC alone). Means of five calves,
target cells: BVD-coated CRBC.
IBR Virus Infection
Clinical observations
Clinical signs in IBR infected calves were usually confined to nasal
and ocular discharges.
Mucous discharges were first observed
on the
second day after inoculation, later became mucopurulent in character and
persisted for at least 6 to 10 days.
There was a tendency for either
larger quantities of discharges, or more persistent discharges, or both,
in calves infected with IBR virus following BVD virus infection; the dif­
ference became evident after the fourth or fifth days following IBR virus
infection, at a time when the discharges started to decrease in calves
infected with IBR virus alone.
Elevations in body temperature were not a constant feature, but four
of the eight calves had increased rectal temperatures (103 F or higher)
on day 3 and 4 following IBR virus inoculation.
Mean temperature readings
78
were approximately the same for calves infected with IBR virus alone or
BVD virus followed by IBR virus infection.
Hematological changes
Total WBCC and B cell counts did not significantly change following
IBR virus infection.
The changes observed in calves infected with both
viruses were similar to those induced by BVD virus alone.
Virus isolation
IBR virus was repeatedly isolated from nasal secretions during more
than 2 weeks following initial infection.
virus shedding by all eight calves.
Table 7 shows a detail of the
A more prolonged excretion was ob­
served in the calves infected with both BVD and IBR viruses.
Response of lymphocytes in culture ^ mitogens following IBR virus infec­
tion
The response of lymphocytes to PHA and PWM were essentially normal
following IBR virus infection.
Responses to PHA and PWM in calves in­
fected with both BVD and IBR viruses followed essentially the same pattern
as in calves inutuldlcu with SVD viriis alcr.s.
Production of circulating interferon
Peak interferon activity was detected in the serum of IBR infected
calves on day 3 after infection (Figure 4) and persisted at decreasing
levels until day 9 in some of the calves.
No differences in serum inter­
feron titers were observed between calves infected with either IBR virus
alone or the combined BVD-IBR virus infection.
Specific iirenune responses
Figure 8 shows the temporal development of antibody to IBR virus.
Neutralizing antibody was first produced between 6 and 9 days post-
79
Table 7.
Isolation of IBR virus from nasal secretions following infection
of cattle with IBR virus alone or with IBR virus 2 days after
challenge with BVD virus
Days following IBR virus infection
Animal
4
6
9
11
+
+
_c
15
18
30
-
ND^
ND
+
+
ND
ND
IBR*
330
331
+
+
335
+
+
-
-
-
-
-
337
+
+
+
+
-
-
+
333
+
+
+
+
+
ND
ND
347
+
+
+
+
+
ND
ND
341
+
+
+
+
+
+
-
NT
+
+
+
-
+
+
-
BVD-IBR®
^Calves infected with IBR virus alone.
'^Positive isolation.
IBR virus identified by FA test.
^Negative.
"Not done.
®Calves infected with IBR virus 2 days after challenge with BVD
virus.
inoculation, reached peak titers on day 17, and decreased thereafter.
Complement greatly increased the neutralizing activity of serum during the
first 3 weeks following infection.
Maximum differences between mean
80
DAYS FOLLOWING IBR INOCULATION
Figure 8.
Development of complement-dependent (C) and complement-inde­
pendent (HC) IBR neutralizing antibody in serum of cattle
following inoculation with IBR virus. Results are expressed as
mean antibody titers (reciprocal of the highest dilution of
serum neutralizing 80% or more of the viral cytopathic effect)
in a micro-neutralization test.
81
titers of complement-dependent and independent antibody were observed on
day 17.
Mean antibody titers to IBR virus were significantly higher in calves
that received the combined BVD-IBR exposure than in calves infected with
IBR virus alone (Figures 9 and 10).
The mean peak antibody titers on day
17 following IBR virus infection were 168 (complement-dependent) and 34
(complement-independent) in calves infected with both BVD and IBR viruses,
as compared to 50 (complement-dependent) and 12 (complement-independent)
in calves infected with IBR virus alone.
In vitro lymphocyte stimulation by IBR virus was first observed on
day 5 following IBR virus infection; responses peaked at day 6, and
dropped to almost background levels between days 11 and 16 (Figure 11).
When the calves were infected 2 days earlier with BVD virus, however,
there was a delay in the appearance of the peak stimulatory activity until
after day 6.
When the mean stimulation ratios of the two groups were com­
pared, they were significantly different on day 6 (student's t test,
n .A m \
n\
Similar differences were observed in the production of interferon by
antigen stimulated lymphocytes; iiraiune production of interferon by lympho­
cyte cultures from calves infected with IBR virus alone was first observed
at day 5.
No interferon was detected in lymphocyte cultures from BVD-IBR
virus infected calves on that day, and the mean interferon titer on day 6
was only one-fourth of the mean titer in calves infected with IBR virus
alone (Figure 5).
The interferon produced by antigen stimulated lympho­
cytes was completely sensitive to pH 2 (Table 3) but showed appreciable
stability at pH 3.
82
256
128
>-
BVD-ÎBR
o
o
00
<
IBR
I—<
u.
o
ht—I
h-
0
10
20
30
DAYS FOLLOWING IBR INOCULATION
Figure 9.
Complement-independent IBR neutralizing antibody in serum of
cattle inoculated with IBR virus alone (IBR) or with IBR virus
2 days after challenge with BVD virus (BVD-IBR).
83
256
128
>•
o
Q
64 -
CO
l-H
h-
BVD-ÎBR
C£
00
H-1
IBR
oc
LU
h"
H
0
20
30
DAYS FOLLOWING IBR INOCULATION
Figure 10.
Complement-dependent IBR neutralizing antibody in serum of
cattle inoculated with IBR virus alone (IBR) or with IBR virus
virus 2 days after challenge with BVD virus (BVD-IBR).
84
4r
0£
in
<
oe
z
o
00
J
I
4
6
8
10
12
14
16
18
20
DAYS FOLLOWING IB-R INOCULATION
Figure 11.
Lymphocyte stimulation by IBR antigen in the period following
inoculation of cattle with IBR virus alone (IBR) or IBR virus
2 days after challenge with BVD virus (BVD-IBR). The stimula­
tion was measured by the incorporation of ^H-thymidine. Re­
sults are expressed as mean stimulation ratios (cpm in IBR
stimulated lymphocytes/cpm in unstimulated lymphocytes).
85
Development of cytotoxic activity jhi peripheral blood leukocytes after IBR
virus infection
Cytotoxic activity also developed after IBR virus infection.
Figure
12 shows percentage of sicr release from IBR-coated CRBC incubated with
lymphocytes collected from calves infected with IBR virus alone or BVD and
IBR viruses.
The activity was in general lower than that observed after
BVD virus infections, but it was specific initially in calves infected
with IBR virus alone.
Table 8 shows the cytotoxic activity of lymphocytes collected at 6, 9
and 11 days postinoculation against IBR-, BVD- or rhinopneumonitis viruscoated CRBC.
The activity was entirely specific on day 6, and still
specific in three of the calves on day 9, although all calves showed non­
specific cytotoxicity by day 11.
The cytotoxic activity of lymphocytes
from those calves infected with both viruses was nonspecific at all times.
The early (day 6) cytotoxic activity was largely retained after
cotton filtration of lymphocytes from calves Infected with IBR virus
aione; the nonadhereni cen population rstainsd full cytotoxic activity in
one-half of the cases.
At this time essentially no activity could be
demonstrated with adherent cells (Table 9).
86
BVD-IBR
lu
if)
<
lU
-J
LU
ac
DC
\T
u
I—'
in
IBR
4
5
6
7
8
9
10
11
18
DAYS FOLLOWING IBR INOCULATION
Figure 12.
Development of cytotoxic activity in peripheral blood leuko­
cytes of cattle following inoculation with IBR virus alone
(IBR) or with IBR virus 2 days after challenge with BVD virus
(BVD-IBR). Results are expressed as mean percentages of sicr
release ± standard error from IBR-coated chicken erythrocytes.
87
Table 8.
Specificity of the cytotoxic activity of peripheral blood leuko­
cytes from cattle inoculated with IBR virus
6 DPI*
9 DPI
11 DPI
IBR
BVD
ERV
IBR
BVD
ERV
IBR
BVD
ERV
330
3.5C
0.8
0.6
6.0
7.8
6.5
1.1
1.7
1.3
331
2.8
0.0
0.0
1.0
0.0
0.2
3.5
1.0
1.2
335
0.9
0.0
0.0
1.2
0.0
0.0
9.9
3.4
1.9
337
1.9
0.0
0.0
1.8
0.0
0.0
4.8
5.4
3.7
333
8.5
5.3
6.2
1.5
1.9
8.5
5.7
8.2
347
14.8
9.8
9.6
3.6
2.9
2.4
5.5
3.2
0.9
341
2.1
2.6
1.8
2.0
1.9
0.9
1.5
0.8
0.4
NT
3.0
3.3
2.6
3.1
3.3
1.3
0.8
1.2
0.7
Animal
IBR^
BVO-IBR^
OC
00
®Days postinoculation.
"C2:ves infected yith IBR
Hlnnê.
Percentage of sicr release (% s^Cr release in the presence of leuko­
cytes - % spontaneous s^Cr release by CRBC alone). Target cells: CRBC
coated with IBR, BVD or ERV antigens.
^Calves infected with IBR virus 2 days after challenge with BVD
virus.
88
Table 9.
Cytotoxic activity of peripheral blood leukocytes on day 6
following inoculation with IBR virus. Identification of
effector eel1 s
Cell populations
Animal
Ficoll-Hypaque
Cotton
Nonadherent
Adherent
330
3.5*
2.9
3.0
0.5
331
2.8
1.4
4.2
0.0
335
0.8
0.5
0.0
0.0
337
1.9
1.1
0.0
0.0
Percentage of sirr release (% ®^Cr release in the presence of leuko­
cytes - % spontaneous s^Cr release by CRBC alone). Target cells: IBRcoated CRBC.
89
DISCUSSION
Recovery from Infection
Most of the studies on immunologic responses to viral infections have
largely assessed the significance of these responses in terms of resist­
ance to reinfection but less attention has been paid to the role of the
immunologic response in recovery from primary viral infections.
The
coincidence in time between recovery and appearance of nonspecific defense
mechanisms, cell-mediated immunity or circulating antibody provides some
indication of the nature of the mechanism promoting recovery.
Attempts at
passive protection of the animal by providing antiviral factors is another
possible approach to the study of recovery.
It is likely that the host
defenses will play different roles in recovery from various viral infec­
tions and, in order to assess the effectiveness of the various antiviral
factors, temporal and spatial considerations are important.
The present study has shown that circulating interferon appears early
in response to BVD virus infection, Jiid it could play an
recovery.
rnlw in
Baron (1970; 1973) has pointed out that interferon, because of
its early appearance^ is an important part of the body's nonspecific re­
actions, probably one of the major contributors to recovery from many
established viral infections.
Circulating interferon is almost always
induced by viruses capable of producing viremia (Baron et al., 1966a).
When passively transferred interferon or noninfective interferon inducers
are injected shortly before infectious virus, the spread of the virus from
blood to target organs is impaired and the survival rates of injected
animals are increased (Baron et al., 1966b; Pinter, 1973).
90
Assessment of the relative role of defense mechanisms in recovery
from BVD infections presents a major problem because of the difficulty in
experimental production of clinical disease with obvious signs of illness
and the sparsity of positive virus isolations.
In this study, however,
lymphocyte responsiveness to mitogens and total WBCC following infection
could be considered as indicators of recovery.
Therefore, the temporal
relationship between appearance of the different defense mechanisms and
the return to normality of these functions can be established.
Circu­
lating interferon was already present 24 hrs after infection and the titer
peaked at day 4.
Lymphocyte responses to PHA and PWM were minimal on day
3, but active responses were evident on day 4.
The lowest blood leukocyte
counts were recorded on days 4 and 5 but rising counts were evident by day
7.
Furthermore, the bacteremia observed after BVD infection, as will be
discussed later, was clearly declining on days 4 and 5, and nonexistent
by day 6.
In comparison, neutralizing antibody, a measure of the specific
immunologic response, was not detected until after day 7.
tions clearly suggest a pruLeuLivc role for intcrferoû.
These observa­
Or. the ether
hand, interferon was not detected after reinoculation of BVD virus into
"immune" animals, a situation in which the protective role of circulating
antibody is evident.
Prior specific immunity is perhaps one possible
explanation for Rosenquist's failure (Rosenquist, 1973) to detect inter­
feron production in most calves infected with BVD virus.
Although the appearance of circulating antibody may not account for
functional recovery of the infected calves, specific antibodies very
likely play a role in the elimination of the virus and termination of the
infection.
In particular, the early complement-requiring antibody
91
response might suggest that complement fixing antibody could play a role
in destruction of infected cells.
This is substantiated by the observa­
tion that BVD virus-infected cells are susceptible to lysis by antiviral
antibody and complement (Ritchie and Fernelius, 1969).
The failure to detect any evidence of a cell-mediated iimune response
agrees with previous reports on the ability of BVD virus to replicate in
"immune" lymphocytes (Truitt and Shechmeister, 1973).
It also agrees with
unpublished observations at the University of Minnesota (C. Muscoplatt,
personal communication) in which there was a failure to demonstrate any
significant degree of stimulation by BVD antigens of lymphocytes from
calves recovering from BVD virus infections.
The possibility of "toxic"
or otherwise inadequate antigens cannot be excluded, however.
Indeed,
Graziano et al. (1975) reported successful lymphocyte blastogenesis by
measles complement fixation antigen despite earlier negative reports where
different antigen preparations were employed.
A most intriguing observation in the present study was the develop­
ment of nonspecific cytotoxic activily nieuiaUu L.y peripharcl bleed Icukccytes following BVD virus infection.
Attempts to locate the activity in
association with different cell populations clearly identified the plasticadherent macrophages as the cy1;otoxic cells.
The increased "killer"
function of macrophages in response to infection is one of the main char­
acteristics of the "activated" macrophage usually associated with recovery
from bacterial infection (North, 1974; Nelson, 1972).
Evidence is accumu­
lating however for a similar role of macrophages in response to viral in­
factions.
Blander (1970» 1971a; 1971b) demonstrated that macrophages play
a major role in recovery from sctrcmslia virus infection in mice.
Blanden
92
and Mims (1973) demonstrated macrophage activation 8 days following infec­
tion with ectromelia or lymphocytic choriomeningitis virus in mice.
Pathak et al. (1974) reported activated macrophages with antiviral and
antibacterial properties following infection of chickens with fowl pox
vi rus.
The general consensus is that macrophage activation occurs following
a cell-mediated immunologic response (Mackaness, 1971; Nelson, 1972;
North, 1974), but such a reaction was not demonstrated during BVD infec­
tion.
Although this failure could be accounted for by inadequate experi­
mental techniques, it is important to bear in mind that there were dif­
ferences in the cytotoxic activity observed following BVD and IBR virus
infections.
The former was completely nonspecific at all times and
mediated by adherent cells.
The latter was specific initially and medi­
ated by nonadherent cells.
This suggests that macrophage activation did
not follow lymphocyte sensitization but was evoked by a different mecha­
nism.
Otîiêr reports ir.dicatG that ccll-ssdiated immunity may
sole means by which macrophages are inactivated.
nnf. hA
thé
Takeya et al. (1968)
observed activated macrophages in neonatally thymectomized mice, and
Blanden and Langman (reported by Blanden and Mims, 1973) made a similar
observation in adult mice, thymectomized as adults, lethal ly irradiated
and reconstituted with isogeneic bone marrow.
These animals had a greatly
depressed capacity for a cell-mediated immunologic response.
Although an
alternate mechanism of macrophage activation is unknown, Alexander and
Evans (1971) reported activation of macrophages, both in vivo and in
vitro, by viral double stranded RNA.
North (1974) observed a great
93
increase in macrophage spreading in vitro after binding of antibody on the
macrophage membrane.
Both mechanisms, if these observations can be con­
firmed and extended to viral infection in vivo> may offer a basis for the
understanding of mechanisms of macrophage activation in the absence of a
cell-mediated immunologic response.
Although normal macrophages are able
to attach to chemically treated erythrocytes (Rabinovitch, 1970), evidence
from the experiments would definitely indicate that there was activation
of plastic adherent cells.
Indeed this is not unlikely since the bac­
teremia associated with BVD virus infection could have been a contributing
factor to the activation process.
Whatever the mechanism of activation, the role of macrophages in re­
covery, if any, is unknown.
Their activity was first recorded 7 days
post-BVD virus infection when recovery was completed. However, activated
macrophages may have been active in the animals long before in vitro
cytotoxicity was detected since in vitro cytotoxic assays may not be ade­
quately sensitive to detect low level in vivo macrophage activity.
In any
event, activated macrophages have been reported to be essential in the
destruction of viral "foci" in ectromelia virus-infected mice (Blandens
1971b) , and presumably they collaborated in the elimination of the BVD
virus infection in my experimental cattle.
The IBR virus infection was essentially localized to the respiratory
tract, both from the clinical and the virological standpoints, since
viremia was not detected at any time.
No attempt was made in this study
to evaluate local defense mechanisms and therefore any conclusion on
mechanisms of recovery from the infection must be based solely on observa­
tions on generalized immunologic responses and reports in the literature.
Circulating interferon was the first observed response with peak
activity occurring on day 3, at least 2 days before specific immunologic
responses were observed.
Interferon is usually produced locally after
intranasal IBR infection (Todd, 1973) and, in view of the relatively high
titers of serum interferon detected, we can assume that such a response
occurred in the present study as well.
Although local production could be
important in protection of susceptible cells, the function of circulating
interferon, other than as a possible indicator of local reactions, remains
largely a matter of conjecture.
If we accept serum interferon as a re­
flection of local production, we must conclude that interferon, although
of possible defensive value early in the disease process, was not a major
factor in clinical recovery from IBR virus infection.
This conclusion is
based on the observation that calves infected with IBR virus alone, or
with both BVD and IBR viruses, produced essentially identical levels of
circulating interferon.
Furthermore, calves previously infected with BVD
virus showed more prolonged virus excretion and more severe mucosal dis­
charges following IBR infection which would indicate a failure OT bvu
produced interferon to provide protection.
The specific immunologic response to IBR virus appeared later than
interferon in the process of infection.
Cell-mediated immunologic re­
sponses were first detected on day 5 following infection.
blastogenic response peaked between days 6 and 9.
The lymphocyte
Although the response
was limited, its temporal development paralleled the specific lymphocyte
transformation pattern reported by Rouse and Babiuk (1974a) following
intranasal infection of calves with IBR virus.
The minimal responses
could be due to the lack of purification of the antigen used in this
95
study, to the age of the experimental animals, or both.
Age differences
are particularly important since very young calves were used in the pre­
vious reports.
The production of "immune" interferon in vitro by stimulatej lympho­
cytes was first detected at day 5 and remained high for several days, with
the highest mean titer recorded on day 11.
The "inrnune" character of the
interferon produced by lymphocytes was concluded from its total suscepti­
bility to pH 2 treatment although still conserving significant activity
after dialysis at pH 3, in agreement with previous renorts (Falcoff,
1972).
The more prolonged production of "immune" interferon as compared
with lymphocyte transformation responses is in agreement with a previous
report on lack of correlation between these responses by human lymphocytes
stimulated with vaccinia virus antigen (Epstein et al., 1972).
The antibody response to IBR virus was detected later than the sixth
day following infection.
The bulk of this early antibody response was
composed of complement-dependent neutralizing antibody, as already repurLeu by Rossi âiiu Kiesel (1374) and Pctglctcr (Î975}.
Humoral antibo^y
is not likely to have been an important factor in the initial events of
recovery due to its late appearance, and because the higher titers were
actually present in the calves infected with both BVD and IBR viruses
where recovery was slower.
The cell-mediated immunologic response on the other hand, was the
only defense mechanism significantly impaired in this group of calves,
where a delay in the peak responses, or overall lower responses were ob­
served.
Rouse and Babiuk (1974a,b) and Davies and Carmichael (1973) have
already suggested a major role for cell-mediated reactions in recovery
96
from ÎBR infection.
They based their conclusions on time of appearance of
the reaction as compared to clinical recovery and analogy with other
herpes viruses known to spread from cell to cell through intercellular
bridges (Christian and Ludovici, 1971) before antibody plus complement can
lyse infected cells (Lodmell et al., 1973).
The present observation of
correlation between delayed cell-mediated responses and prolonged recovery
lends strong support to that view.
The unexpected observation of much higher antibody titers, and espe­
cially complement fixing antibody, in those calves preinfected with BVD
virus raises the possibility of antibody-mediated cell lysis as an immunopathological mechanism hindering recovery (Notkins, 1971).
This possi­
bility cannot be seriously considered, however, without actual determina­
tion of antibody levels in respiratory secretions since different immuno­
globulin classes are involved in the local responses of cattle to respira­
tory viruses (Porter, 1973; Todd, 1973),
Furthermore, although increased
antibody response at the local level could account for more extensive cell
destruction, a more proloiiyeû vit us excrétiori is difficult tc expiai:
since free virus should be rapidly neutralized (Lodmell et al.» 1973).
The cytotoxic activity in the peripheral blood lymphocytes was first
detected on day 6 following IBR infection.
The initial specific activity
associated with nonadherent small lymphocytes, is undoubtedly part of the
cell-mediated immunologic response and very likely has an important role
in recovery by destroying virus-infected cells (Rouse and Babiuk, 1974b;
Lodmell et al., 1973).
The increasing nonspecificity of the reaction with
time after infection suggests the participation of activated macrophages.
97
Although the mechanism of activation is unknown, specifically stimulated
lymphocytes could have been at least partially responsible (Blanden and
Mims, 1973).
The role of these activated macrophages in recovery from ÎBR virus
infection is unknown.
They appeared rather late (9 days) in calves in­
fected with IBR virus alone.
However, nonspecific cytotoxicity was al­
ready apparent at day 6 in calves previously infected with BVD virus and
these calves were slower to recover.
This observation apparently argues
against a protective role of activated macrophages, although a clear
answer to this problem is not possible from the findings of the present
study.
However, a possible role in resistance to reinfection by other
viruses cannot be ruled out.
During the course of the preliminary experi­
mentation conducted prior to research reported in this dissertation,
attempts were made to infect calves from the Veterinary Medical Research
Institute with I BR virus.
virus.
These calves were completely refractory to the
They had been inoculated with respiratory syncytial virus 4-5
weeks previously and, although no other parameters of nonspccific resist­
ance were investigated, it was observed that all of them exhibited sub­
stantial degrees of cytotoxic activity associated with blood leukocytes
prior to the IBR virus challenge.
Similar nonspecific cytotoxic activity
was found in blood leukocytes of several cattle brought to the Department
of Veterinary Clinical Sciences for treatment of different clinical con­
ditions and where exposure to infectious agents is very likely.
These
observations are obviously inconclusive, but it is very tempting to specu­
late that macrophage activation could be a common reaction of cattle to
98
infection and that such cells, at least in the case of IBR virus, could
play a role in resistance to infection.
Effect of BVD Virus Infection on the Immune System
Despite the repeated observation of BVD virus involvement in multi­
ple-etiology disease conditions, the only experimental evidence for a
possible iimiunosuppressive effect has come from indirect studies.
Truitt
and Shechmevster (1973) reported that BVD virus can infect both macro­
phages and lymphocytes under in vitro conditions.
Muscoplatt et al.
(1973a) observed inhibition of lymphocyte responses to PHA after in vitro
infection or during the course of experimental disease.
The present study has confirmed some of these results such as the
observation of a sharp reduction in lymphocyte responsiveness to PHA and
PWM during the first 3 or 4 days following infection.
PHA responses are
usually not dependent on macrophage function (Waldron et al., 1973) and
therefore BVD virus must act by a direct inhibitory effect on T lympho­
cytes .
At! increase in the ûuabcr of ccVls
surface inwiynncjinhiilin was
also observed particularly on day 2 following infection.
This increase
was most likely a relative one due to a decrease in other cell popula­
tions, but became less pronounced as the total number of white cells de­
creased.
This could be interpreted as a preferential or more dramatic
effect of the virus on the nonimmunoglobulin bearing cells, thereby im­
plying the possibility of a relative resistance of B cells to the leuko­
penic effects of infection.
Reports of alteration in Immunoglobulin
synthesis in chronic BVD infection (Muscoplatt et al., 1973b) as evidenced
by a decreased number of circulating B lymphocytes or lack of antibody
99
response to BVD virus are somewhat contradictory with the present work.
However, the depressed antibody response in chronic BVD infections could
be a cause rather than a consequence of the infection.
There is no evi­
dence to support a primary viral etiology and the possibility of a primary
immunodeficiency has not been investigated (Malmquist, 1968; Bittle and
House, 1973),
The more direct evidence for an immunosuppressive effect came from
the observation of bacteremia and the study of the immunologic response to
IBR virus.
The occurrence of positive blood cultures in up to 85% of in­
fected calves, with consistently negative cultures from control or reinoculated "immune" calves, is a strong indication of a causal relation­
ship between BVD infection and bacteremia.
It clearly indicates a nega­
tive effect of infection on normal defense mechanisms and, more particu­
larly, the blood clearance function associated with tissue and circulating
macrophages (Raffel, 1951).
The positive cultures from normal calves recorded when medium with
SHb was used indicates that bacteremia is of cunmiun uLcurrenCc in cattle.
The use of BPS, known to be anticomplementary and to inhibit the normal
bactericidal action of blood and the phagocytic activity of leukocytes
(Ellner and Stoessel, 1966), apparently allowed the isolation of bacteria
normally inhibited when regular blood culture medium was used.
A concept
of "normal" bacteria in the circulation of cattle should not be sur­
prising.
In man, commensal microorganisms from the normal flora of the
mouth, nasopharynx and intestines can be carried from time to time into
the blood where they are normally promptly phagocytized (Joklik and Smith,
1972).
In cattle, pathogenic microorganisms, and more notably CI. chauovei
100
and Cl. novyi, are known to invade the body through enterohepatic circula­
tion and can be isolated from normal livers or spleens (Smith and
Holdeman, 1968).
The uninhibited presence of bacteria in blood during infection would
be best explained by multiplication of the virus in macrophages with re­
sultant cell death or functional impairment.
Probably a variety of bac­
terial organisms were present in the blood of the animals but the almost
exclusive isolation of Bacillus spp. was a reflection of the experimental
procedures; these microorganisms could have inhibited the growth of other
bacteria because of rapid multiplication with resulting competition or by
production of antibiotics (Korzybski et al., 1967).
Bacillus cereus, repeatedly isolated from infected or normal calves
in this experiment, has been incriminated in bovine abortions (Wohlgemuth
et al., 1972a).
Small doses of the organism, when given i.v. to pregnant
cows, were apparently cleared from the blood before fetal infection
occurred; large doses were necessary to overcome the reticuloendothelial
system (Wohlgemuth et al,, 1972b;.
BVD viriis infectiGr. could be ens
possible mechanism to facilitate placental colonization by Bacillus
C6r6U5.
Macrophages are also an important nonspecific mechanism of defense
against some viral infections (Mims, 1964).
It is possible that if this
bacteremia was actually due to a suppression of macrophage function, this
alteration could have been an important factor in the prolonged recovery
from I BR infection in experiments 5 and 6.
The present study does not
101
offer an answer to this question since no evaluation was made of macro­
phage function in the respiratory tract where the IBR virus infection was
localized.
The almost complete inhibition of lymphocyte responses to T cell
mitogens during the leukopenic phase of BVD infection suggests that T cell
functions were impaired during infection.
T cell function is specifically
altered in a number of other viral infections including measles
(McFarland, 1974b), influenza (Kantzler et al., 1974), lactic dehydrogenase
virus (Howard et al., 1969) and lymphocytic choriomeningitis virus (BroJfSrgensen et al., 1975).
Temporary suppression of T cell function by BVD
virus is a likely mechanism for the delayed cell-mediated responses to IBR
virus.
The mechanism is unknown, but BVD virus replication in T lympho­
cytes could result in death, functional alteration or reduced expansion of
a specific population of T cells.
Loss of T cell function could also
implicate loss of the normal regulatory functions of T cells in antibody
production (Gershon, 1974; Katz and Benacerraff, 1972; Baker et al., 1974)
uiierKU_y yruv i u i iiy a iiicviian i siii lui une mt^icaocu unuiuuujr i ut mu >< • un .
Alternatively, the mechanism of immunosuppression could reside in a
loss of macrophage function,
Soontiè'ns and Van der Veen (1973) observed
that the inhibition of PHA responses in human lymphocyte cultures infected
with poliovirus is due to a direct action on the macrophages.
According
to Hall and Kantor (1972) the depression of pre-existing delayed hyper­
sensitivity during mumps virus infection is due to an inhibition of the
nonspecific mononuclear effector cells.
However, adherent cells are
necessary for optimal antibody production to complex antigens ( Greaves
et al., 1973) and the idea of an altered macrophage function would be
102
difficult to conciliate with increased antibody production.
Furthermore,
even if BVD virus inhibits tissue and circulating macrophages as suggested
by the appearance of bacteremia, there is no conclusive proof that the
adherent cells involved in the induction of immunologic response are
phagocytic macrophages (Nelson, 1972).
At least in the immunologic re­
sponse of mouse spleen cultures to erythrocyte antigens, there is evidence
now that the macrophages that digest the particulate antigens are distinct
from the adherent cells required to deliver the inductive stimulus to pre­
cursor antibody-forming cells or to thymus-derived helper cells (Watson
et al., 1974).
The reason for a preferential action of BVD virus on T lymphocytes,
with minor or no effects on B lymphocytes, is not clear.
Woodruff and
Woodruff (1974) have reported the presence in mouse T lymphocytes of
specific receptors for several myxo- and paramyxoviruses such as in­
fluenza, Newcastle and Sendai viruses which are known to cause lymphopenia
or to alter blastogenic transformation of lymphocytes.
Whether or not
similar receptors exist in taille, or even their sigr.lficsr.cc ir. viral
immunosuppression, is unknown.
In conclusion, BVD virus seems to exert an immunosuppressive effect
by action at two levels:
(1) nonspecific defense mechanisms, presumably
the phagocytic cells; and (2) the specific cell-mediated immunologic re­
sponses by interfering with T cell function.
Such a combination of
effects is potentially very significant in the pathogenesis of many viral
or bacterial infections, and may explain a role for BVD virus infection in
multiple etiology syndromes of cattle.
103
SUMMARY
Specific and nonspecific mechanisms of recovery from viral infection
were studied in feeder calves after intravenous challenge with BVD virus,
intranasal challenge with IBR virus or a combination of both viruses.
BVD virus infection resulted in inhibition of the normal blood
clearance mechanisms as evidenced by the detection of an endogenous bac­
teremia in up to 85% of infected calves on the first 5 days following in­
fection.
The responses of lymphocytes in culture to PHA and PWM were
drastically reduced on the first 4 days following inoculation.
Relative
increases in the number of peripheral blood 3-lymphocytes during the
leukopenic phase of the disease suggested a preferential action of the
virus on T lymphocytes.
Circulating interferon was consistently de­
tectable after BVD virus infection.
Peak titers occurred at the same time
that responses to mitogens, white blood cell counts and blood clearance
mechanisms started to regain pre-inoculation levels.
Antibody production
was not detected prior to the eighth day following infection, when all the
above functions had returned to normal.
No specific cell-mediated immu­
nologic responses were elicited by BVD virus infection.
BVD virus infection 2 days before IBR virus challenge resulted in a
delayed recovery from IBR infection as evidenced by a more protracted
clinical condition and a more prolonged period of virus excretiorî.
The
production of circulating interferon was not affected and the titers of
antibody to IBR virus were increased after BVD virus infection.
In con­
trast, cell-mediated immunologic responses to IBR virus, as measured by
104
specific lymphocyte transformation and production of interferon by antigen
stimulated lymphocytes, were significantly delayed.
It was concluded that, while circulating interferon may have played a
major role in recovery from BVD virus infection, cell-mediated immunologic
responses correlated much more closely with clinical recovery from IBR
virus infection.
An immunosuppressive role of BVD virus infection was demonstrated.
The effects of the virus were apparently exerted on at least two mecha­
nisms, normal blood clearance mechanisms and T-cell function.
Macrophage activation was demonstrated after infection by both
viruses with the appearance of cytotoxic activity mediated by peripheral
blood leukocytes.
Plastic adherent cells nonspecifically lysed chicken
erythrocytes coated with viral antigens.
105
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ACKNOWLEDGMENTS
I am deeply grateful to Dr. Merlin L. Kaeberle, my graduate advisor,
for his guidance and encouragement throughout this study and his help in
the preparation of this manuscript.
I also wish to thank Drs. V. A. Seaton, F. K. Ramsey, M. S. Hofstad,
J. Robyt and R. E. Dierks, the other members of my graduate committee,
for their time and advice.
The technical assistance of Mrs. Katie Einck, Miss Dawn Mangold, Mr.
R. Lindahl and Mr. R. Johnson, as well as the assistance and kindness of
the entire staff of the Veterinary Diagnostic Laboratory and the Depart­
ment of Veterinary Microbiology and Preventive Medicine, are greatly
appreciated.
The author wishes to recognize the contribution of Jensen-Salsbery
Laboratories (Kansas City, Kansas) to this investigation by procuring the
experimental animals and funding the experimentation.
The Iowa Beef Industry Council provided the funds for the construc­
tion of the animal facilities without which this investigation would not
have been possible.
I thank Ms. Charmian Nickey for excellent typing assistance.
My greatest expression of gratitude is to my wife Cristina and our
children Carolina and Juan Pablo for their paticncc and encouragement
during the course of my graduate training.