Evaluation of the cell-mediated immune response to bovine respiratory syncytial virus infection in
cattle
by Emery Waine Field
A thesis submitted in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE
in Veterinary Science
Montana State University
© Copyright by Emery Waine Field (1979)
Abstract:
Six Holstein calves were experimentally infected by intranasal instillation with 7.5 X 10^7 plaque
forming units (PFU) of bovine respiratory syncytial virus (BRSV), strain VC-494. One calf was
maintained as a noninfected control. The calves were evaluated for the development of a cell-mediated
immune (CMI) response using the leukocyte migration inhibition (LMI) test under agarose and the
delayed hypersensitivity skin test (DHST). Serological evaluations were conducted prior to infection
and at the conclusion of the study using the indirect fluorescent antibody test (IFAT).
Results of the LMI test indicate that the infected calves did develop a CMI response which was
detected as early as five days postinfection. The response, measured as migration inhibition, reached a
maximum value of 34%, 21 days after the experimental infection. An enhanced inhibition, which
developed more rapidly, was observed with leukocytes from one calf reinfected with BRSV.
DHS tests were conducted following the LMI evaluations. Infected calves developed intradermal
reactions which reached maximal intensity between 48 and 72 hours. The control calf did not develop a
delayed type of reaction. Results of the IFAT showed that serum antibody levels also increased in the
calves following the experimental BRSV infection.
Results of this study indicated that both humoral and cell-mediated immune responses were elicited in
the calves infected with BRSV, strain VC-494. STATEMENT OF PERMISSION TO COPY
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requirements fo r an advanced degree a t Montana State U n iv e rs ity ,
I agree th a t the Library shall make i t fre e ly a v a ila b le fo r in ­
spection.
I fu rth e r agree th a t permission fo r extensive copying
of th is thesis fo r scholarly purposes may be granted by my m ajor.
professor, o r, in his absence, by the D irecto r of L ib ra rie s .
It
is understood th a t any copying or publication of th is thesis fo r
fin a n c ia l gain shall not be allowed without my w ritte n permission
Signature
Date
Qaxeut^ r .
EVALUATION OF THE CELL-MEDIATED IMMUNE RESPONSE
TO BOVINE RESPIRATORY SYNCYTIAL VIRUS
INFECTION IN CATTLE
by
EMERY WAINE FIELD
A thesis submitted in p a r tia l f u lfillm e n t
of the requirements fo r the degree
of
MASTER OF SCIENCE
in
V eterinary Science
Approved:
Graduate ©ean
MONTANA STATE UNIVERSITY
Bozeman, Montana
August, 1979
■ ■
y1
V
Iii
ACKNOWLEDGEMENTS
The author would lik e to extend his sincere appreciation to
Dr. M. H. Smith fo r his support and encouragement throughout this
study.
He would also lik e to thank his graduate committee fo r th e ir
contributions and advice with his research p ro je c t.
A special thanks
go to Sandra P h illip s and Donna Gollehon fo r th e ir e x c e lle n t technical
assistance with th is p ro je c t.
Thanks also go to his w ife Deanna and son C lin t 9 fo r th e ir
patience and understanding during his study a t Montana State Univer­
s ity .
TABLE OF CONTENTS
LIST OF TABLES...........................................................................' .
.
.
.
LIST OF F IG U R E S .............................................................. ......
v
vi
CHAPTER
INTRODUCTION
.................................................................................
Statement o f Purpose ..............................................................
2.
LITERATURE REVIEW . .
C haracteristics o f the Virus
Human RSV ...............................
Bovine RSV
.....................................
3.
4.
I
2
CTl
4=»
1.
.
.
.
.
.
.
.
12
MATERIALS AND M E T H O D S ................................
29
Experimental Animals ...........................................
Virus and V ira l Antigen ....................................................... . 2
In d ire c t Fluorescent Antibody Test
...............
Delayed H yp ersen sitivity Skin Test
. . . . . . .
Leukocyte M igration In h ib itio n Test
...............
RESULTS .............................................................
C lin ic a l Observations .
Leukocyte Migration In h ib itio n Test .
belayed H yp ersen sitivity Skin Test
In d ire c t Fluorescent Antibody Test
.
.
29
9
31
32
34
37
37
. . .
37
...............
45
....................46
5.
DISCUSSION
.......................................................
6.
SUMMARY........................................... .................................... ......
48
.
55
V
LIST OF TABLES
Table
Page
1.
Maximum P ostinfection Tem peratures............................... .
2.
LMI Test with BRSV A n t i g e n ............................................................... 39
3.
' LMI Test with BRSV and IB R /P I-3 Antigen
.
37
.
.
45
r-
4.
Delayed H yp ersen sitivity Skin T e s t .....................................................46
5.
Serological Response
....................................................................
;
47
vi
LIST OF FIGURES
Figure
.,.I.
-2 .
3.
Page
, LMI M igration P a t t e r n ........................................................
Mean LMI Response
......................... .
LMI Response A fte r Reinfection with BRSV .
.
.
38
.
40-41
43-44
vi i
ABSTRACT
Six Holstein calves were experim entally in fected by jn tranasal
in s t illa t io n with 7.5 X IO^ plaque forming units (PFU) of bovine
re sp irato ry syncytial virus (BRSV), s tra in VC-494. One c a lf was
maintained as a noninfected c o n tro l. The calves were evaluated fo r
the development of a cell-m ediated immune (CMI) response using the
leukocyte m igration in h ib itio n (LMT) te s t under agarose and the delayed
h y p e rs e n s itiv ity skin te s t (DHST). Serological evaluations were con­
ducted p rio r to in fe c tio n and a t the conclusion o f the study using the
in d ire c t fluorescent antibody te s t (IF A T ).
Results of the LMI te s t in d ic a te th a t the in fected calves did
develop a CMI response which was detected as e a rly as fiv e days post­
in fe c tio n . The response, measured as m igration in h ib itio n , reached
a maximum value o f 34%, 21 days a f t e r the experimental in fe c tio n . An
enhanced in h ib itio n , which developed more ra p id ly , was observed with
leukocytes from one c a lf rein fected with BRSV.
DHS tests were conducted follow ing the LMI evaluations. In ­
fected calves developed intradermaI reactions which reached maximal
in te n s ity between 48 and 72 hours. The control c a lf did not develop
a delayed type o f reactio n . Results of the IFAT showed th a t serum
antibody levels also increased in the calves follow ing the experimental
BRSV in fe c tio n .
Results of th is study indicated th a t both humoral and c e llmediated immune responses were e lic it e d in the calves Tnfected with
BRSV, s tra in VC-494.
CHAPTER I
INTRODUCTION
Bovine re s p ira to ry disease (BRD) continues to constitu te a
major economic loss fo r c a ttle producers in the United States (7 1 ).
The widespread use of vaccines against v ir a l and b a c te ria l pathogens
c u rren tly associated with the disease has fa ile d to prevent outbreaks
o f re s p ira to ry in fe c tio n in c a t t le .
This in effectiveness of trie
vaccines suggests th a t other e tio lo g ic agents may be involved in the
pathogenesis of the disease.
The f a ilu r e to prevent outbreaks of BRD
also indicates th a t a complex in te rre la tio n s h ip exists between environ­
mental fa c to rs , the in fectio us agent, and the immune response of the
host animal in determining the outcome o f resp irato ry t r a c t in fe c tio n .
Bovine re s p ira to ry syncytial virus (BRSV) is widespread in
c a ttle and is recognized as a primary e tio lo g ic agent of BRD (9 , 94,
97, 104).
Recent evidence suggests th a t the virus can also occur as
a concurrent in fe c tio n with other v ir a l pathogens including p a ra in flu ­
enza-3 ( P I - 3 ) , in fectio u s bovine rh in o tra c h e itis (IB R ), and bovine
v ir a l diarrhea (BVD) viruses (9 , 6 2 ).
Vaccines have been developed
against P I-3 , !BR, and BVD v ir a l in fec tio n s but an e ffe c tiv e vaccine
against BRSV is unavailable.
The type of v ir a l disease manifested and the s e v e rity , dura­
tion and d is trib u tio n of the disease is in part a re s u lt of the in t e r ­
2
action o f the in fec tio u s agent and the host's defense mechanisms.
These mechanisms are comprised of both s p e c ific and nonspecific fa c ­
to rs .
The nonspecific fa c to rs , usually ac tiv e against a v a rie ty of
viruses, co n stitu te "native immunity", and are fo r the most part a
genetic property of an animal species (8 9 ).
The s p e c ific immune
mechanisms, on the other hand, are dependent upon the host animal's
exposure to a p a rtic u la r v iru s .
There are two general types of
e ffe c to r mechanisms which mediate s p e c ific a lly acquired immunity;
those mediated by c e ll products o f sensitized lymphocytes (humoral
immunity) and those mediated by the lymphocytes themselves and the
c e lls re cru ited by these sensitized lymphocytes (cell-m ediated
immunity).
The humoral immune response o f c a ttle to BRSV in fe c tio n has
been investigated using serological techniques (7 7 ).
The r e la tiv e
importance of th is response in counteracting the in fe c tio n however,
remains undefined.
Cell-m ediated immunity has not been demonstrated
to p a rtic ip a te in the immune response of c a ttle to BRSV in fe c tio n .
A b e tte r understanding of the importance of the immune response of
c a ttle to BRSV in fe c tio n w ill depend upon defining the mechanisms in ­
volved and how various events interphase to combat the in fe c tio n .
Statement of Purpose
The purpose of th is study was: ( I ) to determine i f c e ll-
3
mediated immunity (CMI) is e lic it e d follow ing experimental in fec tio n
of calves with BRSV and (2) i f CMI is e lic it e d , to determine the mini
mal time between exposure to the virus and detection o f sensitized
lymphocytes.
CHAPTER 2
LITERATURE REVIEW
Respiratory syncytial virus (RSV) is recognized as a re s p ir­
atory tr a c t pathogen in man and c a t tle .
Since it s i n i t i a l is o la tio n
from a chimpanzee in 1956, the virus has been shown to be the major
cause of severe re s p ira to ry disease in human infants (5 , 16).
The
virus produces a type o f in fe c tio n and disease unlike th a t of any other
known re sp irato ry tr a c t pathogen (1 4 ).
The f i r s t reported is o la tio n of a bovine re s p ira to ry syncy­
t i a l virus (BRSV) was in Switzerland in 1970 from c a t tle with acute
re sp irato ry disease (8 6 ).
The virus was subsequently shown to be
associated with bovine re sp irato ry disease in several countries and
is now recognized as one of the primary e tio lo g ic agents of the bovine
re s p ira to ry disease complex (4 3 ).
C haracteristics of the Virus
The human and bovine re s p ira to ry syncytial (RS) viruses to­
gether with the pneumonia virus o f mice (PVM) are c la s s ifie d as pneumo­
viruses, members of the paramyxoviridae fam ily (72, 111).
Spherical
forms of both the human and bovine RS viruses range from 80 to 400 nm
in diameter (6 , 48, 49, 54, 84).
Filamentous forms, exceeding 2 urn in
length, have been observed budding from cytoplasmic membranes (8 4 ).
Although there is s t i l l some controversy concerning the dimensions
of the RSV h e lic a l nucleocapsid, i t appears to be approximately 14 nm
5
in diameter with a h e lic a l pitch o f 6.5 nm (6 , 54).
Virus assembly
occurs by budding through virus modified c e ll membranes where the v i r ­
ion acquires it s envelope containing glycoprotein surface projections
(6 , '49, 8 4 ).
The lack o f in h ib itio n by halogenated deoxyribosides
suggests th a t the v ir a l genome consists o f a single stranded RNA (39,
4 8 ).
Buoyant densities fo r both the human and bovine RS viruses have
been determined and range from 1.22 to 1.24 g/ml (21, 4 8 ).
S e n s itiv ity
to low pH has been demonstrated with complete loss o f a c t iv it y a t pH
3.0 (39, 48, 86, 104).
The virus also appears to be r e la tiv e ly thermo-
la b ile , losing 90% of it s in f e c t iv it y when maintained a t 55 C fo r 5
minutes (38, 48).
Unlike other members of the paramyxoviridae fa m ily , neith er
hemagglutination nor neuraminidase a c t iv it y have been demonstrated with
e ith e r virus (9 0 ).
S trains of the virus are not s e ro lo g ic a lly id e n ti­
cal but antigenic v a ria tio n is lim ite d and does not appear to be pro­
gressive (20, 22).
The RS viruses can be grown in c e ll cultu re using conventional
tissue c u ltu re techniques.
Human RS virus has been grown in human can­
cer c e lls including HeLa, Hep 2, and Chang liv e r c e lls (5 5 ).
The
bovine RS virus re p lic a te s in a v a rie ty of bovine c e lls including
kidney, lung, t e s t ic le , a o rta , and rectum c e lls (86, 66, 104).
In c e ll c u ltu re , the primary cytopathic e ffe c t is the forma­
6
tio n of m ulti nucleated e p ith e lia l gian t c e lls termed syncytia..
These
syncytia, developing w ith in 5 to 6 days p o s tin fe c tio n , often contain
numerous eosinophilic cytoplasmic inclusion bodies (55, 86, 1Q4).
Human RSV
In 1956, Morris and associates (81) isolated an agent from
a chimpanzee with an upper re s p ira to ry t r a c t in fe c tio n .
This newly
discovered agent, an RNA v iru s , was shown to be s e ro lo g ic a lly d is tin c t
from previously id e n tifie d paramyxoviruses and was designated chimpan­
zee coryza agent (CCA).
An e tio lo g ic association was established
between th is is o la te and a re sp irato ry illn e s s observed in a laboratory
worker who had been in close contact with the chimpanzee.
In the
follow ing year Chanock and co-workers, attempting to recover new agents
from infants with severe lower re s p ira to ry tr a c t illn e s s , reported two
is o la tio n s of a s im ila r virus from children in Baltimore (1 8 ).
Using
n e u tra liz a tio n te s ts , the virus was shown to be s e ro lo g ic a lly indis-k
tinquishable from the CCA.
Cell cultures infected with the virus
developed a syncytial type of cytopathic e ffe c t.
Owing to it s assoc­
ia tio n with re s p ira to ry illn e s s and the fa c t th a t i t induced the forma­
tio n of syncytia in c e ll c u ltu re , the virus was designated resp irato ry
syncytial virus (RSV).
Serological surveys have shown th a t RSV in fectio ns are quite
common in the human population.
Over 70% of the persons tested poss-r
essed n e u tra liz in g antibodies fo r the virus by fiv e years of age and
7
over 90% by 15 years of age (12, 15, 3 7 ).
The lowest incidence of
n e u tra liz in g antibody was found in the. 6-month to one-year age group
which is temporally associated with the disappearance o f passively
acquired maternal antibody. .
Annual epidemics of RSV in fectio n s occur p rim a rily from la te
f a l l to e a rly spring, with the peak months being January and February.
These epidemics are associated with a dramatic increase in the number
of in fan ts and young children requiring h o s p ita liz a tio n fo r lower
re s p ira to ry t r a c t disease (15, 5 7 ).
This is in contrast to the v i r ­
tual absence o f confirmed RSV in fectio n s during the summer months.
May through August.
Since it s i n i t i a l is o la tio n , RSV has emerged as the major res­
p ira to ry tr a c t pathogen o f infancy and e a rly childhood (5 , 10, 16, 25,
H O ).
The virus is also considered to be a major cause o f fa ta l res­
p ira to ry tr a c t disease in the f i r s t year of l i f e .
A study in B rita in
has shown th a t 19 of 22 children with f a ta l re sp irato ry tr a c t in fe c ­
tions were under one year o f age and RSV was isolated from eight of
these patients postmortem (3 4 ).
A v a rie ty of c lin ic a l patterns o f resp irato ry illn e s s have been
associated with the is o la tio n of RSV but the most common are brochi o l i t i s and bronchopneumonia (1 5 ).
The virus was shown to be asso­
ciated with re s p ira to ry illn e s s in a large proportion o f infants ad­
m itted to a c h ild re n 's hospital during the f i r s t three months of 1962.
8
The RSV illn esses ranged from mild upper re sp irato ry tr a c t in fectio n
to severe b ro n c h io litis and bronchopneumonia. A ten month old boy,
from whom RSV was is o la te d , died 24 hours a fte r admission to the hos­
p ita l with severe lower re sp irato ry tr a c t disease.
Microscopic exam­
in ation of re s p ira to ry tissues revealed c e llu la r necrosis of the
tracheobronchial mucosa, focal consolidation of both lungs, and
severe emphysema, suggesting an incomplete bronchial or bronchio la r
obstruction (4 5 ).
The severe disease occurs most often in children one month
to six months of age, the frequency of disease then decreases with
increasing age (16, 17).
Older children and adults usually develop
a less severe disease with predominately an upper re s p ira to ry in ­
volvement (5 2 ).
This a g e -illn e s s re la tio n s h ip of RSV disease is s t i l l '
not completely understood but in vestig ato rs have advanced several
theories.
Infants with the severe disease were found to possess moderate
to high levels of m aternally derived antibody s p e c ific fo r the virus
(8 7 ).
The decreasing le v e ls of these passively acquired antibodies
with increasing age appeared to c o rre la te with the decreasing frequency
of disease.
This apparent c o rre la tio n indicated th a t serum antibody
did not provide e ffe c tiv e protection against the lower resp irato ry
tr a c t disease.
Furthermore, these observations provided the basis fo r
Chanock e t a l . (13) to speculate th a t serum antibody might a c tu a lly
9
contribute to the development of the serious lung damage.
They pro­
posed th a t a type 2 or possibly a type 3 immune h y p e rs en s itiv ity reac­
tion might re s u lt from the in te ra c tio n of p re -e xistin g antibody and
v ir a l antigen in the lungs.
Vaccination studies using a fo rm a lin -in a c tiv a te d , alump re c ip ita te d RSV vaccine appeared to provide support fo r th is ‘; theory
(56, 5 8 ).
Children receiving th is k ille d vaccine developed substan­
t i a l levels of serum n e u tra liz in g antibody to the v iru s .
Upon ex­
posure to a w ild -ty p e RSV however, 80% of the children in the
vaccinated group required h o s p ita liz a tio n and two of the children
died.
This unexpected re s u lt was in terp reted as c le a rly establishing
the involvement of serum antibody in the pathogenesis of the severe
RSV disease (1 3 ).
Recent reports have fa ile d to support th is theory
and i t now appears th a t serum antibody does provide a p rotective e ffe c t
against the severe disease (8 , 6 1 ).
Data from a 10-year study of
re sp irato ry illn e s s in normal children in dicate th a t gradually
accumulating levels of humoral antibody p a ra lle l the acquired re s is ­
tance to RSV in fectio ns (4 0 ).
There is substantial reduction in
the attack ra te in susceptible in divid u als and also in the severity
of illn e s s produced by a th ird re -in fe c tio n with RSV.
These findings
in dicate th a t both age and immunity, although not necessarily serum
antibody, act to am eliorate the illn e s s associated with in fe c tio n due
to RSV.
10
I f serum antibody does not provide adequate protection against
the severe RSV disease, other mechanisms must be involved since older
children and adults do show some resistance. Cell-m ediated immunity .
(CMI) could conceivably play a s ig n ific a n t ro le in lim itin g the c e ll
to c e ll spread of the v ir a l in fe c tio n .
Results obtained from vaccin­
ation t r ia ls and natural RSV in fectio n s in dicate th a t a CMI response ;
does occur but th a t i t does not prevent the development of the serious
disease (59, 98, 99).
The ro le of in te rfe ro n in recovery from RSV in fe c tio n is s t i l l
unclear.
C o n flic tin g resu lts have been obtained regarding the induc­
tion of in te rfe ro n by RSV and it s s e n s itiv ity to the a n tiv ir a l protein
(33, 41, 75).
Although in fan ts appear to be capable o f producing s ig ­
n ific a n t levels of in te rfe ro n , McIntosh (67) detected only low levels
in children with RSV in fe c tio n s .
Hall e t a l. (36) found th a t the
quantity o f in terfe ro n produced by children with RSV in fectio ns was
s ig n ific a n tly less than those with influenza and parainfluenza virus
in fe c tio n s .
Whether these resu lts suggest th a t in te rfe ro n is not in ­
volved in recovery from RSV in fe c tio n or th a t low le vels are adequate
in lim itin g in fe c tio n w ill require fu rth e r in v e s tig a tio n .
Local secretory antibody has been shown to be a major deter­
minant in resistance to v ir a l re s p ira to ry tr a c t in fe c tio n s .
M ills
e t a l . (74) observed th a t high le vels of s p e c ific IgA in nasal secre­
tions prevented RSV in fe c tio n in adult volunteers regardless of the
11
level o f serum antibody. Other, studies have indicated th a t local secre
tory antibody may also provide a c u rative e ffe c t in RSV in fec tio n s .
McIntosh e t a l.
(68) found th a t the disappearance of virus from nasal
secretions correlated with the appearance of secretory antibody.
Naso
pharyngeal e p ith e lia l c e lls from in fan ts with confirmed RSV infections
have been shown to be coated with an anti-RSV IgA (32, .69),. ',,This
coating of the in fected c e ll with s p e c ific antibody could n e u tra lize
v ir a l p a rtic le s as they bud from the surface of the infected c e ll.
Another p o s s ib ility is th a t the antibody prevents the successful mat­
uration of virus a t the c e ll surface, thereby preventing the release
of in fe c tiv e v ir a l progeny.
This type of action has been demonstrated
in influenza A in fectio ns where the antibody prevented the release of
in fe c tiv e v ir a l p a rtic le s (2 4 ).
These studies coupled with the ineffectiveness o f vaccineinduced serum antibody, have encouraged the development o f an attenu­
ated s tra in of RSV which can induce resistance without producing
s ig n ific a n t illn e s s (1 1 ).
A chemical mutagen, 5 -flu o ro u rid in e , has
been used to produce a tem perature-sensitive (ts ) mutant which is
designated ts -1 .
When administered in to the nasopharynx of adult
volunteers, the mutant induced resistance without producing s ig n if i­
cant illn e s s .
I t appeared g e n e tic a lly stable under laboratory con­
d itio n s but When children were infected experim entally with the ts -1
they shed a g e n e tic a lly alte red virus with a w ild -ty p e temperature
12
s e n s itiv ity (4 2 ).
Current research e ffo rts are directed a t producing
a more g e n e tic a lly stable ts mutant o f the RSV.
I t w ill hopefully
provide a means of preventing the severe disease which occurs in in fants less than one year o f age.
Bovine RSV
In 1968, Doggett e t a l . (23) found n e u tra liz in g a c tiv ity to
human RSV in bovine sera suggesting the presence of an anti gem" caI l y
s im ila r agent in c a t tle .
Then in 1970, Paccaud and Jacquier (86) re ­
ported the is o la tio n o f a v ir a l agent from c a ttle in Switzerland.
They isolated the virus from a cow and a c a lf during an outbreak of
resp irato ry disease in February o f 1967.
The disease was characterized
p rim a rily by an upper re sp irato ry tr a c t in fe c tio n with nasal discharge
and a mild cough.
In approximately one th ird of the cases however, a
high fever developed with discrete signs of bronchopneumonia.
The
seroconversion of these c a t tle , in n e u tra liz a tio n and complementfix a tio n te s ts , c le a rly established th a t the is o la te was of bovine
o rig in .
N e u tra liza tio n tests using human sera indicated th a t the v iru s ,
or an a n tig e n ic a lly related agent, was also present in the human pop­
u la tio n .
This fa c t together with c h a rac te ris tic s of the is o la te such
as a c id - la b ilit y , chloroform s e n s itiv ity , and a syncytial type of cytopathic e ffe c t in c e ll c u ltu re , suggested th a t the virus might be a
13
bovine representative of the RSV.
C ro ss-n eu tralizatio n and compIe -
ment fix a tio n tests c le a rly established the close antigenic r e la tio n ­
ship between the is o la te and human RSV.
With th is evidence the authors
concluded th a t the new is o la te was indeed a bovine re s p ira to ry syncy­
t i a l virus (BRSV).
Following th is i n i t i a l is o la tio n Itiaba and associates (46, 47)
reported the is o la tio n of a BRSV from c a ttle during an outbreak of
re sp irato ry disease in Japan.
The disease swept across Japan during
the months from October 1968 to A p ril 1969.
C lin ic a lly i l l c a ttle
exhibited anorexia, depression, pyrexia, re sp irato ry d is tre s s , and
a mild cough.
In fa ta l cases, emphysema and consolidation of the
lungs were noted.
The close antigenic re la tio n s h ip between this virus
and human RSV was established using n e u tra liz a tio n te s ts .
data indicated th a t the virus was widespread in Japan.
Serological
Over 60% of
the c a ttle tested possessed detectable levels of n e u tra liz in g a n t i­
bodies to the v iru s .
Experimental in fe c tio n of a c a lf with the new
is o la te produced pyrexia, anorexia, depression, serous nasal discharge,
and a leukopenia.
The c a lf developed n e u tra lizin g antibody against
the virus which were detectable two weeks postinoculation.
In 1971, Wellemans reported a BRSV is o la te from c a ttle with
resp irato ry disease in Belgium (112, 113).
The virus was described
as the p rin c ip le v ir a l e tio lo g ic agent associated with bovine re s p ir­
atory disease in th a t country.
E ffo rts were in it ia t e d to develop an
14
attenuated s tra in of BRSV which could be used in a vaccination program
against the v ir a l re la ted disease.
Jacobs and Edington (50, 51) reported the is o la tio n of a BRSV
from a group of 14 calves in Dorset, England.
The calves had a serous
nasal discharge, pyrexia, increased re sp iratio n r a te , and a mild cough.
The is o la tio n confirmed serological data th a t indicated the virus was
prevalent throughout B rita in .
Experimental in fec tio n o f gnotobiotic,
colostrum-deprived, and conventional calves produced a temperature
response with a serous nasal discharge in a ll three experimental groups.
The virus was re -is o la te d from nasal secretions of in fected calves four
to ten days postinoculation.
The f i r s t is o la tio n o f BRSV in the United States was reported
by Smith, Frey, and Dierks in 1974 (1 0 3 ).
They is o la te d the virus
from Iowa fe e d lo t c a ttle with an acute resp irato ry disease.
Biological
and physical c h a ra c te ris tic s together with c ro s s-n eu tra liza tio n tests
indicated a close but non-identical re la tio n s h ip to human RSV (104).
Serological data indicated th a t 81% of the c a ttle from 43 herds pos­
sessed serum antibody to the v iru s .
Experimental in fe c tio n of fiv e
calves with the is o la te produced c lin ic a l illn e s s characterized by
anorexia, pyrexia, leukopenia, nasal discharge, and m alaise.
The
virus appeared capable o f producing illn e s s in calves possessing
maternal antibody fo r the v iru s.
15
Rosenquis t , in 1974, reported the is o la tio n o f two strains of
BRSV from nasal secretions of calves with acute re s p ira to ry disease
in Missouri (9 4 ).
The calves exhibited c lin ic a l signs of re sp irato ry
illn e s s , including pyrexia, increased re sp iratio n ra te , and nasal d is ­
charge.
Three calves, out of a group of nine, developed s ig n ific a n t
levels of n e u tra liz in g antibody to the virus is o la te .
Since its i n i t i a l is o la tio n in Iowa, the virus has been shown
to be prevalent in several s ta te s . . P otg ieter and Aldridge (8 8 ), using
the in d ire c t fluorescent antibody te s t (IFAT) to detect BRSV s p e c ific
antibody, reported th a t 73% of the cows from 160 herds in Oklahoma
were seropositive fo r BRSV.
In three herds in which resp irato ry d is ­
ease developed, the incidence of BRSV seroconversion approached 100%.
Mohanty e t a l . (76) conducted a serological survey of n e u tra lizin g
antibody to BRSV in M aryland.: T h irty -e ig h t of 100 c a ttle surveyed
possessed antibody to the v iru s .
In a recent serological survey
of Montana c a ttle a t Montana State U n iv e rsity, Smith (personal com­
munication) demonstrated widespread exposure to the v iru s .
These
studies in dicate th a t BRSV may be as prevalent in c a ttle as human RSV
has been shown to be in the human population.
In the f a l l when calves are weaned, they are exposed to physi­
cal stress and are freq uently placed in feedlots with calves from
other geographical regions.
These factors often contribute to the
development of resp irato ry disease.
The disease has been referred to
16
as "shipping fever" but new terminology designates the illn e s s as
"bovine re s p ira to ry disease complex".
The present concept of the
e tio lo g y o f re sp irato ry tr a c t disease o f c a ttle is th a t i t is a m u lti­
fa c to ria l syndrome involving stre s s , v i r a l , and b a c te ria l in fectio ns
(4 3 ).
Parainfluenza-3 ( P I - 3 ) , in fectio u s bovine rh in o tra c h e itis (!B R ),
and bovine v ir a l diarrhea (BVD) viruses have been given considerable
sig n ifican ce in the etio lo g y of the disease but l i t t l e
a tten tio n has
been given to the involvement of BRSV.
Lemkuhl and Gough (62) collected serum samples from e arly
weaned Iowa f a l l calves shortly a f t e r the onset of re sp irato ry tr a c t
disease.
Seroconversion ra te of c a ttle to IBR virus was 4.3%, P I-3 -
16.3%, BVD
- 9.6%, and BRSV - 45.4%.
They also observed an increased
rate of seroconversion fo r !BR, P I-3 , and BVD viruses in the presence
of BRSV seroconversion.
These resu lts appear to im plicate BRSV in
th is disease of calves with the p o s s ib ility of BRSV in fe c tio n f a c i l i ­
ta tin g in fe c tio n by other v ir a l pathogens.
In a recent re p o rt, Bryson and associates (9 ) described four
outbreaks of c a lf pneumonia in which there was serological evidence
of concurrent in fectio ns with P I-3 and BRS viruses.
CeUves exhibited
reduced ap p etites, nasal discharge, coughing, dyspnea, and pyrexia.
An emphysematous crackling was also noted over the diaphramatic lobes
of the lungs.
Three calves out of a group of six fa ile d to respond to
a n tib io tic treatment and died w ithin fiv e days of the onset of disease.
17
Postmortem examination of these calves revealed pneumonia
involving the cran ial lobes and severe pulmonary emphysema.
Purulent
exudate was evident in the bronchioles and there were areas of in t r a ­
lo bular hemorrhage.
Microscopic examination revealed the most severe
lesions were those in the bronchioles, resp irato ry a c in i, and small
blood vessels.
Bronchitis and b ro n c h io litis were evident, with a f f e c t ­
ed airways containing an exudate of polymorphs, mononuclear c e lls , and
desquamated e p ith e lia l c e lls .
A lveolar lesions consisted of over-
in fla te d a lv e o li, areas of a lv e o la r collapse, focal necrosis, a lv e o la r
edema, and congestion of the a lv e o la r blood vessels.
M ultinucleated
e p ith e lia l syncytia were prominent in bronchio lar and a lv e o la r walls
with intracytoplasm ic inclusion bodies w ith in these syncytia.
Whether BRSV was t o t a lly responsible fo r the lesions or acted
in concert with P I-3 cannot be determined but the involvement of
BRSV in the development of these lesions is most c e rta in ly im plicated.
Immunological Response to BRSV In fec tio n
Natural and experimental BRSV in fectio ns in c a ttle have been
shown to e l i c i t a humoral response with the development of detectable
levels of both nasal secretory and serum antibody (47, 51, 77, 82, 94,
104, 113).
The r e la tiv e importance of these antibodies in the re s is ­
tance to the v ir a l in fe c tio n , or recovery from i t , has not been c le a rly
established.
Serum antibody does not appear to play a prominent ro le in
18
resistance to v ir a l re s p ira to ry tr a c t in fe c tio n s .
Mohanty e t aI . (77)
observed th a t calves challenged in tra n a s a lIy with BRSV developed
c lin ic a l illn e s s in the presence or absence o f c irc u la tin g antibody.
Smith e t a l . (104) also reported th a t calves with p re -e x is tin g serum .
: i t
antibodies fo r BRSV developed c lin ic a l illn e s s upon intranasal chalIenge with the v iru s .
S im ila r re su lts have also been reported in
studies with P I-3 virus in fec tio n of calves.
Levels o f serum a n ti­
body did not c o rrelate with resistance to in fec tio n but did appear
to be associated with reducing the s e ve rity of disease (2 7 ).
These studies in c a ttle are in agreement with the results
of human RSV studies where the level of serum antibody fa ile d to
co rrelate with resistance to re s p ira to ry in fec tio n (1 3 , 8 7 ).
These
studies also in dicate th a t serum antibody appears to be involved
in reducing the s e v e rity of re s p ira to ry illn e s s .
Serum antibody plays a major ro le in preventing those diseases
in which the virus must trav e l through the blood stream to reach
it s ta rg e t organ.
In re sp irato ry t r a c t in fections where the ta rg e t
organ is also the portal of e n try , local factors including in te rfe ro n ,
secretory antibody, and local cell-m ediated immunity appear to be the .
most important defense mechanisms (9 3 ).
A secretory antibody system has been id e n tifie d in the bovine
species with IgA being the predominant immunoglobulin in nasal and
lacrim al secretions (6 4 ).
This secretory antibody system can function
19
independently from the systemic antibody system and can be activated
by local application of antigen- (96, 1.09).
This fa c t is of extreme
importance where the primary s ite of virus re p lic a tio n is the re s p ira ­
tory tr a c t .
Studies of v ir a l re sp irato ry in fec tio n in man have shown
th a t the presence of antibody in nasal secretions correlates with
protection against in flu e n za , p arainflu enza, and RSV in fectio n s ( I ,
21, 65, 74, 95, 102).
.
.
.
Studies with P I-3 virus in fe c tio n in c a ttle also indicate th a t
local secretory antibody (IgA)
confers b e tte r protection against res­
p ira to ry tr a c t in fe c tio n than c irc u la tin g antibody (2 7 , 3 5 ).
Mohanty
e t a l . (77) reported th a t four of fiv e calves inoculated i/ntranasally
with BRSV developed detectable levels o f nasal secretory antibody.
When these calves were re-challenged with the virus fiv e weeks a fte r
the primary challenge, they were found to be s o lid ly immune.
This was
in terpreted as evidence of the importance of secretory antibody in
resistance to BRSV in fe c tio n .
I t should be noted however, th a t a non­
s p e c ific fa c to r, possibly in te rfe ro n , may have been responsible fo r
the observed immunity in these calves.
These studies, w hile not c le a r­
ly establishing secretory antibody as the p rotective mechanism in re ­
sistance to re sp irato ry v ir a l in fe c tio n , strongly im plicate the
p a rtic ip a tio n of antibody in the process.
In focusing our a tten tio n on these s p e c ific a lly acquired
immune responses, i e . , the serum and secretory antibody responses, the
20
importance o f nonspecific mechanisms of local protection should not
be overlooked.
Factors such as m u co cilliary b a rrie rs , temperature,
and nonspecific phagocytosis by a lv e o la r macrophages play a s ig n if icant ro le in resistance to v ir a l in fe c tio n .
In a d d itio n , nonspecific
e ffe c ts of in fe c tio n , such as c e llu la r resistance due to in terfero n
production, may often determine the fin a l outcome o f the disease (7 3 ).
Once a v ir a l in fe c tio n is established, with virus re p lic a tio n
occurring in a f u l ly susceptible host, active defense mechanisms must
be e lic it e d to combat the in fe c tio n .
As described e a r lie r in th is
review, viruses th a t promote systemic infections with viremia are
controlled p rim a rily by c irc u la tin g antibody.
On the other hand,
viruses which spread from c e ll to c e ll via in te r c e llu la r bridges and
which usually elaborate s p e c ific antigens on the c e ll surface, are
more lik e ly to e l i c i t a CMI response (85, 115).
Examples o f th is type
of virus include members of the orthomyxo- and paramyxoviridae fa m ilie s .
Most of the members o f these fa m ilie s have been shown to e l i c i t both
cell-m ediated and humoral immune responses (2 8 ).
The CM! response can occur lo c a lly in the re s p ira to ry tr a c t
a fte r intranasal in fe c tio n without a generalized response, or a t le a s t
with a diminished one (78, 112).
T-lymphocytes, stim ulated by contact
with v ir a l antigen on the surface of infected c e lls , may become d ir ­
e c tly cytotoxic to the infected c e lls .
These cytotoxic reactions are
very lik e ly to be important in the control of noncytolytic v ir a l
21
in fectio ns (115).
The in te ra c tio n between these !-lymphocytes and
the infected c e lls can also re s u lt in the release of soluble media­
tors c a lle d lymphokines.
The bio lo g ical a c tiv ity of lymphokines
a ffe c t the behavior o f macrophages, polymorphonuclear leukocytes,
lymphocytes, and other c e ll types in v i t r o .
A l i s t o f these factors
published in a report of a World Health Organization s c ie n tific group
(115) is found in ta b le I .
The follow ing is an excerpt from R.E. R ocklin's chapter on
mediators of c e llu la r immunity (2 9 ).
I t is .a q u a lita tiv e in te rp re ­
ta tio n of the events th a t may occur in certain cell-m ediated reactions
based upon the existence o f these lymphokines.
----- " A ntigen-sensitive lymphocytes, when stim ulated
by the appropriate antigen, become activated and s ta rt
synthesizing the various lymphocyte mediators.
Chemo-
ta c tic factors fo r monocytes and polymorphonuclear
leukocytes re c ru it inflammatory c e lls to the reaction
s ite .
Once th e re , the macrophages and polymorpho­
nuclear leukocytes might be held a t the s ite by MIF
and LIF.
Macrophages might be activated to an en­
hanced sta te by the action of the a c tiv atin g fa c to r.
Other lymphocytes are re cru ited to p a rtic ip a te in the
reaction by the mitogenic fa c to r.
Lymphocyte mitogen­
ic fa c to r nonspecificalIy activates the other lym­
phocytes in the area which would perhaps, in tu rn ,
s ta r t producing lymphocyte mediators.
These events
have the e ffe c t of am plifying an i n i t i a l l y small
reactio n.
Once a c tiv a te d , the inflammatory c e lls
22
Table I .
A.
B iological a c t iv it ie s of products of activated lymphocytes9
A ffectin g macrophages
.
Migration in h ib itio n fa c to r
(MIF)
in h ib its the m igration of
normal macrophages
Macrophage aggregation fa c to r
(MAF)
agglutinates macrophages
in suspension
Macrophage chemota c tic fa c to r
(MCF)
cause macrophages to
migrate through micropore
f i l t e r along gradient
Macrophage resistance fa c to r
(postulated)
renders macrophages nons p e c ific a lIy re s is te n t to
in fe c tio n with certain bac
te r ia and viruses
C ytophilic antibodies
confer on macrophages
s p e c ific r e a c tiv ity with
antigen
B. A ffec tin g lymphocytes
Blastogenic or mitogenic fa c to r
(BF
or
MF)
induces b la s t c e ll trans­
formation and t r it ia t e d
thymidine incorporation
in normal lymphocytes
P otentiating Factor
(PF)
augments or enhances on­
going transform ation in
mixed lymphocyte culture
or antigen-stim ulated
cultures
Cell cooperation or helper fa c to r
produced by T c e lls , in ­
creases the number or rate
of formation of Ab-producing c e lls in v itro
Suppressor fa c to r (postulated)
in h ib its a c tiv a tio n o f,
and/or antibody produc­
tio n by B c e lls
23
Table I .
C.
( C o n t i n u e d ) _____
A ffec tin g granulocytes
In h ib itio n fa c to r
(L IF )
causes granulocytes to
migrate through micro­
pore f i l t e r along a
gradient
Chemotactic fa c to r
D.
in h ib its the m igration of
human buffy coat c e lls or
peripheral blood leuko­
cytes from c a p illa ry tubes
or wells in agar plates
A ffectin g cultured c e lls
Lymphotoxin
( L I)
cytotoxic fo r certain
cultured c e lls , e . g . ,
mouse L c e lls or HeLa
cells
P ro life ra tio n in h ib itio n fa c to r
and cloning in h ib itio n fa c to r
(P IF ,
CLIF)
in h ib it p ro life ra tio n of
cultured c e lls without ,
lysing them
protects c e lls against
virus in fe c tio n
In terfero n
E.
Producing e ffe c ts in vivo
Skin re active fa c to r (possibly
a combination, of several of
the above a c t iv it ie s )
Macrophage disappearance fa c to r
(SRF)
in normal guinea pig skin
induces indurated skin re ­
actions th a t are s im ilar
h is to lo g ic a lly to delayed
h y p e rs en s itiv ity reactions
in jected in tra p e r ito n e a lly , causes macrophages
to adhere to peritoneal
wal I
aFrom: Cell-m ediated immunity and resistance to in fe c tio n . World
Health Organization Technical Report Series No. 519. 1973. WHO,
Geneva.
24
become b a c te ric id a l or tu m o ric id al.
Furthermore, the
vasoactive properties of some of the mediators may
account fo r p art o f the inflammation.
Other protein
systems, including the complement system, the kinin
system, and the c lo ttin g system are also c a lle d into
play.
I f antigen remained a t the s it e , such a reac­
tio n would continue, abating only as the antigen
supply was exhausted."
This in te rp re ta tio n is purely speculative since i t has not as y e t
been possible to confirm in v iv o .
The apparent c o rrelatio n between the in te ra c tio n , in v it r o , of
sensitized lymphocytes and appropriate antigens, and delayed hyper­
s e n s itiv ity skin reactio ns, led to the establishment o f a series of in
v i tro correlates of cell-m ediated immunity.
These in v itr o correlates
have been extensively d e ta ile d and reviewed by Bloom and Glade ( 7 ) ,
the World Health Organization (1 1 5 ), and McCluskey and Cohen (7 0 ).
Therefore, consideration of th is subject w ill be lim ite d , with emphasis
on the two techniques th a t re la te d ire c tly to the experimentation
described in th is th e s is .
D elayed.H ypersensitivity Skin Test
When c e rta in antigens are in je c te d into the skin of sensitized
animals, an inflammatory response may develop a t the in je c tio n s ite .
Erythema and swelling gradually appear, reaching maximal in te n s ity from
24 to 72 hours a fte r the in je c tio n .
H is to lo g ic a lly the reaction is
25
characterized by the accumulation o f large numbers of macrophages
and lymphocytes.
This response to the antigen can be tran sferred from sensitized
'
to normal animals through the use of lymphocytes, but not byxmeans of
serum, in d icatin g th a t the response is cell-m ediated.
The reaction is
the re s u lt of the in te ra c tio n of sensitized lymphocytes and appropriate
antigen.
These c irc u la tin g , a n tig en -sen sitive Tr>lymph:ocytesv; upon
encountering.the in jected antigen respond by both re c ru itin g other
lymphocytes and by d iv id in g , d iffe r e n tia tin g , and releasing lymphokines.
Some o f these lymphokines, chemotactic factors and m igration in h ib itio n
fa c to rs , are thought to be responsible fo r the accumulation of the
large number of macrophages and lymphocytes.
Vascular changes are
mediated through the release of "sk in -re a c tiv e factors" as well as by
the release o f lysosomal enzymes from the macrophages.
As the macro­
phages ingest and eventually destroy the antigen, the stimulus fo r
fu rth e r lymphokine production dim inishes, perm itting the tissues to
return to normal (108).
. Skin te s tin g with the appropriate antigen is the most impor­
tan t diagnostic te s t fo r CMI (115).
Perhaps the best known example
of th is te s t is the Mantoux re ac tio n , obtained by in je c tin g tuber­
culin antigen into the skin of a sensitized animal.
In c a t tle , the
caudal fo ld is usually used as the in je c tio n s it e , with po sitive
animals developing the c h a ra c te ris tic erythema and indurated swelling
I
26
from 24 to 48 hours la t e r .
Although a neck s ite has been shown to be
more s e n sitive the d if f ic u lt y in re stra in in g the animal make the caudal
fold the most commonly used in je c tio n s ite in c a ttle .
Other antigens
including mumps, P I-3 , histoplasm osis, and toxoplasmosis have been
used fo r assessing delayed h y p e rs e n s itiv ity to in fectio us diseases in
man and animals (79, 115).
Leukocyte M igration In h ib itio n (LMI) Test
_
.
In 1967 Soborg and Bendixen (106) described an in v itr o te s t
based upon the in h ib itio n of the m igration of human p e rip h e ra l. leuko­
cytes from glass c a p illa ry tubes.
They found th a t th is s p e c ific in ­
h ib itio n correlated with the presence o f delayed h y p e rs en s itiv ity to
brucella antigen.
In recent years, th is or m odifications o f the t e s t,
have been applied in a number of studies of c e llu la r immunity.- The
method is now recognized as an in v itr o correlate of c e llu la r immunity
in man and animals (.105, 106).
In 1971 Clausen (19) developed a leukocyte m igration in h ib itio n
(LMI) te s t in which the leukocytes migrate out from w ells punched in
an agarose g e l.
The leukocytes, p rim a rily polymorphonuclear neutro­
phils (PMN), migrate out from the w ells between the agarose and the
surface of the p e tri dish, forming a c irc u la r area of migrated c e lls .
The migration is in h ib ite d i f appropriate antigen is preincubated with
the antig en -sen sitive lymphocytes.
These lymphocytes in te ra c t with the
antigen and produce lymphokines, including a leukocyte in h ib ito ry
2Z
fa c to r ( L IF ) .
This LIF has been shown to be chemically d is tin c t from
the m igration in h ib ito ry fa c to r (MIF) th a t in h ib its the m igration of
macrophages in the macrophage m igration in h ib itio n te s t (9 1 ).
The
presence of c y to p h ilic antibody, antigen-antibody complexes, and anti
gen alone do not a ffe c t the m igration patterns in the LMI te s t (4 4 ).
Erard (26) demonstrated the s u it a b ilit y of the LMI te s t in
agarose fo r routine detection of CMI in man.
He found a good corre­
la tio n between the PPD skin te s t and the in h ib itio n of leukocyte
m igration using th is antigen.
Other investigators have employed the
te s t to evaluate c e llu la r immunity to numerous antigens including
toxoplasmosis (3 1 ), h e p a titis B (2 6 ), and Candida antigen (4 ).
Anders and Natvig (71) used the LMI te s t to measure CMI to mumps,
herpes simplex, adenovirus, and cytomegalovirus antigen.
They found
a good c o rrelatio n between the resu lts o f the LMI te s t and skin
r e a c tiv ity to the antigen.
In 1977 Moreno-Lopez evaluated the CMl response of c a ttle to
P I-3 virus in fe c tio n .
Test systems included the skin-hypersensi­
t i v i t y (SH) t e s t, lymphocyte stim ulation (LS) te s t, and the leukocyte
m igration in h ib itio n (LMI) te s t.
In four calves vaccinated in tra -
nasal Iy with P I-3 v iru s , in h ib itio n o f leukocyte m igration was
observed two weeks a fte r vaccination.
Maximal in h ib itio n reached
50 to 60% o f control values, and occurred ten weeks p o stin fectio n .
Results of the LMI te s t correlated with those obtained with both the
28
SH and LS te s ts .
The LMI te s t was described as being more rapid to
perform and more reproducible when compared to the LS te s t (8 0 ).
In a recent review o f in v itr o methods in CMI, the LMI te s t
in agarose was characterized as being easy to perform, ra p id , and a
r e lia b le c o rre la te of cell-m ediated immunity in man and animals (107).
’
■/
‘
v
•
/I
.
1: f 1
*• .
i ,
: :•
v;
The te s t requires only a. minimal amount of:'antigen which can. be very
crucial when only small amounts of p u rifie d antigen are a v ailab le (26)
The te s t offers an e x c e lle n t tool in evaluating the c e llu la r immune
response to a large number o f agents in both man and animals.
CHAPTER 3
MATERIALS AND METHODS
Experimental Animals
Seven H olstein calves, three to eigh t months o f age, were
purchased from local farms fo r use in th is study.
Two of the calves,
approximately three months of age, were seronegative fo r BRSV.
The
remaining fiv e calves possessed low levels o f serum antibody fo r the
virus as determined by the in d ire c t fluorescent antibody te s t (IFAT)
( 88).
7
Six calves were inoculated in tra n a s a lIy with 7.5 X 10
forming units (PFU) of BRSV/celI suspension.
plaque
One of the calves was .
also inoculated in tra n a s a lly with four m i l l i l i t e r s o f IB R /P I-3 modi­
fie d liv e virus vaccine.
The remaining c a lf was housed separately
and was kept as a noninfected contoI .
Virus and V ira l Antigen
Bovine RSV, s tra in VC-494, was propagated in a continuous
c e ll lin e of Georgia bovine kidney (GBK) c e lls .
Media fo r growth and
maintenance of c e ll cultures consisted o f Dulbecco's medium 1 supple­
mented with 10% fe ta l c a lf serum 1 (FCS) and adjusted to pH 7.4.
Infected c e ll cultures were incubated a t 37 C fo r 72 - 96 hours
and harvested when 70 - 80% of the monolayer showed a cytopathic
1G r a n d I s l a n d
B io lo g ic a l
C o . , G rand
Is la n d ,
New Y o r k .
30
e ffe c t (CPE).
The cultures were then subjected to three cycles o f
freeze-thawing followed by sonication fo r 20 seconds a t a se ttin g of
35 kc/second.
C e llu la r debris was p a r t ia lIy removed by low speed, c e n trifu ­
gation (1000 X g) fo r 20 minutes.
PFU titr a tio n s were then performed,
the amount of virus present ranging from 1.5 X 10^ to 5 .0 X 10® PFU/ ml.
The v ir a l suspension was aliquoted in to appropriate amounts and frozen
a t - 70 C.
The BRSV te s t antigen was prepared from GBK c e ll cultures in ­
fected with s tra in VC-494.
The infected c e ll cultures were frozen
at - 70 C when .70 - 80% of the c e ll monolayer
showed CPE.
The
v ir u s /c e ll suspension was then allowed to thaw a t 37 C and p a r t ia lly
p u rifie d and concentrated according to the procedure described by
S e n te rfit and Baldridge (100).
B r ie f ly , c e ll debris was p a r t ia lly
removed by low speed c e n trifu g a tio n (1000 X g) fo r 20 minutes.
The
virus in the re s u ltin g supernatant f lu id was then concentrated by
p re c ip ita tio n with 6% (w/v) polyethylene g lyco l-6000
and p a r t ia lly
p u rifie d by sucrose-gradient ce n trifu g a tio n fo r 16 hours a t 105,000 X g.
The re s u ltin g band of v ir a l antigen was recovered and dialyzed against
fiv e changes of 50 volumes of phosphate-buffered sa lin e solution
(PBSS; pH 7 .4 ) a t 4 C.
The concentration of v ir a l protein was deter­
mined according to the method of Lowry e t a l. (6 3 ).
I
S ig m a C h e m i c a l
C o .,
S t.
L o u is , M is s o u ri.
A control antigen
31
was prepared from noninfected c e ll cultures according to the procedure
described above.
An IB R /P I-3 modified liv e virus vaccine
■i
was used to inoculate
one o f the BRSV in fected calves in th is study. A d ilu tio n of th is vac­
cine was also used as a te s t antigen in the LMI te s t.
Selection of Optimum Protein Concentration
The optimum protein concentration of the BRSV te s t antigen
preparation was established by a dose-response study.
A sample of the
te s t antigen was adjusted to 10, 25, 50, 100, 150, 200, and 250 ug/ml
of protein .
The d ilu tio n of the antigen which in h ib ite d sensitized
leukocytes with minimal e ffe c t upon nonsensitized leukocytes was d e te r­
mined.
The antigen preparation was d ilu te d to a protein concentration
of 50 ug/ml and stored a t - 70 C in 0 .1 ml aliquots u n til needed.
The
control antigen was treated s im ila r ly and stored u n til used in the
LMI te s t.
I n d irec t Fluorescent Antibody Test (IFAT)
'
The IFAT was performed according to the procedure of Potgieter
and
Aldridge (8 8 ).
Cells were grown in four-w ell chamber slides
infected with BRSV, s tra in VC-494.
p
and
A fte r 20 hours of incubation, the1
2
1
Jensen-Salsbery L a b ., Kansas C ity , Missouri.
2
Lab-Tek Products, D ivision Miles Lab. In c ., Kankakee, I I .
32
c e lls were washed with PBSS and fix e d in acetone fo r ten minutes.
The.
slides were stored a t - 70 C u n til needed.
The te s t was performed by rehydrating the slid es in a moist
chamber a t room temperature.
Ten-fold d ilu tio n s of serum samples, in ­
cluding known negative and p o s itiv e samples, were placed onto the in fected c e lls .
The slid es were then incubated fo r 30 minutes a t 37 C.,
Following three washings in PBSS (pH 7 .5 ) the slid es were a ir dried.
An anti-b ovine IgG conjugate
,. d ilu te d 1:16 in PBSS, was placed onto
the slides and allowed to incubate fo r 30 minutes.
This incubation was
followed by three washings in PBSS, a f t e r which the slides were allowed
to a ir dry.
Coverslips were mounted using Well come1s medium (pH 8 .9 ) .
The slides were examined using a microscope equipped with a
d a rk fie ld condenser and a 200-W mercury vapor lamp.
The in te n s ity of.
the fluorescence was rated su b jec tive ly from 0 (negative) to a +4
(very in ten se).
Delayed H yp ersen sitivity Skin Test
Calves were in je c te d intraderm alIy into a shaved area on the
la te r a l portion o f the neck with 0.2 ml o f the BRSV antigen and with
0.2 ml of the v iru s -fre e control antigen.
The distance between the'
in je c tio n s ite s was approximately 10 cm.
The thickness of the skin­
fo ld a t the s ite of each in je c tio n was measured using a micrometer
i
Miles L a b ., I n c . , Research d iv is io n , E lk h a rt, In .
33
gauge s h o rtly before and a t 24, 48, and 72 hours a f t e r the in je c tio n
of antigen.
The in je c tio n s ite s were also checked a t s ix to eight
hours to insure th a t an Arthus type o f reaction Was not in te rfe rin g
with the la t t e r evaluations.
Preparation o f Peripheral Blood Leukocytes
Peripheral blood leukocytes were iso lated using a m odification
of the method described by Naylor and L i t t l e (8 3 ).
Blood (20 ml) was
I
collected in a 20 cc syringe containing preservative fre e heparin
(10 lU /m l) .
The blood was divided equally in to two centrifuge tubes,
each containing 20 ml of d is t ille d water.
A fte r shaking gently fo r
45 to 60 seconds, 10 ml of 0.0132 M phosphate b u ffe r in 2.7% NaCl solu­
tion (pH 6 .8 ) was added to restore is o to n ic ity .
centrifuged fo r ten minutes a t 450 X g.
The tubes were then
The supernatant f lu id was
discarded and the p e lle ts of white c e lls were washed once in Hank's
i
BSS
containing heparin (5 lU /m l) followed by two washings in the same
medium but without heparin.
The c e lls were then resuspended in TC med­
ium Parker 199 * supplemented with 10% PCS, p e n ic illin (60 lU /m l) and
streptomycin (60 ug/ml)
per m i l l i l i t e r .
I
to a concentration of 2 - 2.5 X 10
Q
cells
The v ia b i lit y of the c e lls was found to be greater
than 90%. by trypan blue exclusion.
Average d if fe r e n tia l count was
34% n e u tro p h ilic granulocytes, 56% lymphocytes, 7% monocytes, and
^G rand
Is la n d
B io lo g ic a l
C o . , G rand
Is la n d ,
New Y o r k .
34
3% eosino philic granulocytes.
Preparation of Agar Medium fo r the LMI Test
The agarose plates were prepared using a m odification of the
procedure described by Gaines e t a l . (3 1 ).
A 1.6% agarose suspension
was mixed with FCS and TC medium Parker. 199 supplemented with p e n ic il­
lin (60 lU /m l) and streptomycin (60 ug/m l).
was 0.8% agar and 10% FCS.
The fin a l concentration
Six m i l l i l i t e r s of the agar medium was
poured in to disposable p la s tic p e tri dishes
(60 X 15 mm).
The agar
medium was allowed to cool a t room temperature and was then placed in
a cooler a t 4 C fo r 15 to 30 minutes.
Six w e lls , 3 mm in diameter,
were punched in the agarose in each dish and the plugs were gently
removed by suction.
The dishes were then incubated a t 37 C in an
atmosphere of 2% CO^ in a ir u n til use.
The pH of the agarose medium
was between 7.2 and 7.5 .
Leukocyte M igration In h ib itio n Test
A m odification of the procedure described by Clausen (19) was
used.
The BRSV antigen was prepared as described above.
Aliquots
(0 .2 ml) of the leukocyte suspension were incubated fo r 45 minutes a t
37 C with aliquots (0 .1 ml) o f the BRSV te s t antigen and the control
antigen.
i
The c e lls were resuspended a t ten minute in te rv a ls during
Lux S c ie n tific Corporation, Newbury Park, C a lifo rn ia .
35
incubation to f a c i l i t a t e maximal contact with the antigen.
Following
th is incubation period 10 ul of the mixture of c e lls and BRSV antigen
or control antigen were placed in to each w e ll.
Each dish contained
three wells with c e lls and BRSV antigen and three wells with c e lls plus
control antigen.
The plates were incubated a t 37 C in an atmosphere
of 2% COg in a ir enclosed w ithin a moist chamber.
A fte r 18 hours o f incubation the c e lls were fix e d .in the agarose dish with 8.0% glutaraldehyde
1
f o r 30 minutes.
removed and the c e lls stained with crystal v io le t.
The agar was then
The. c e lls were
rinsed twice with d is t ille d water and then allowed to a ir dry.
The degree of. c e ll migration was determined by measuring the
lin e a r distance of m igration from the edge of the w ell to the perimeter
of the area covered by the migrating c e lls .
For th is purpose a micro­
meter disc was used and the values expressed as the number of grids
covered by m igrating c e lls .
The m igration in h ib itio n in percent was
calculated fo r each te s t well according to the formula:
^ _ Mean lin e a r m igration o f antigen treated c e lls
x
Mean lin e a r m igration o f control c e lls
An average of three re p lic a te s of each te s t was used in the fin a l
computations.
IS ig m a
C h e m ic a l
C o ., S t.
L o u is , M is s o u r i.
36
Cytological Examination
C haracterization of the migrated c e lls was accomplished by
stainin g the c e lls a fte r 18 to 20 hours of incubation.
A covers!ip
( 2 2 X 40 mm) was placed in the bottom of the plate before the addition
of the agarose medium.
described.
The p late was then treated as previously
At the end of the incubation period the plate was flo ate d
on water (45 C) fo r 15 minutes.
The agarose medium was then c a re fu lly
removed and the covers!ip placed in absolute methanol a t room temper­
ature.
The c e lls were stained in W right's stain according to standard
staining procedures.
CHAPTER 4
RESULTS
C lin ic a l Observations
Calves experim entally infected with BRSV developed c lin ic a l
.
illn e s s characterized by nasal and lacrim al discharge, increased res­
p ira tio n r a te , depression, and a tra n s ie n t pyrexia.
Nasal discharge
was f i r s t observed on the f i f t h day po stinfection and persisted fo r
four to fiv e days.
The maximum po stinfection temperatures of in d iv i­
dual calves are shown in table I .
Table I .
C alf No.
Maximum P ostinfection temperatures_________ ________ ,_______
Maximum temperature
Day Postinfection
I
104.6
8
2
105.6
8 .
3
107.2
8
4
104.8
9
5
102.8
6
105.6
:
"
8
. .
8
Average preinfection temperatures ranged from 101.2 to 102.0 F.
Leukocyte Migration In h ib itio n Test
The m igration of bovine peripheral leukocytes from wells punch­
ed in an agarose gel is shown in fig u re I .
The leukocytes migrated
between the agarose layer and the surface of the dish to form a c ir -
38
Figure I .
Cell m igration patterns from wells
containing leukocytes plus BRSV antigen ( T ) , and
from wells containing leukocytes plus v iru s -fre e
control antigen (C ).
cular area of c e lls .
A fte r 18 hours of incubation the m igrating c e lls
formed a c le a r-c u t edge which f a c ilit a t e d accurate measurement.
Microscopic examination of the c e lls indicated th a t the poly­
morphonuclear (PMN) leukocytes were the predominant c e lls involved in
the m igration.
Monocyte m igration did not exceed 60% o f the to ta l l i n ­
ear m igration.
Lymphocytes were found only in close proxim ity to the
w ells.
Six of the calves were infected by intranasal in s t illa t io n with
7.5 X IO^ PFU of BRSV, s tra in VC-494.
a noninfected c o n tro l.
The remaining c a lf was kept as
The resu lts of the LMI te s t are shown in table
39.
2 and fig u re 2.
.
Table 2.
i','.
:
LMI Test with BRSV Antigen*
Days P ostinfection
-7
0
5
7
14
21
28
35
42
4
5
11
20
34
39
32
27
29
2
I
13
18
27
31
23
29
ND
3
8
. 14
28
27
34
35
32
32
26
4
11
13
23
36
30
37
37
31
25
5
8
6
27
33
32
30
32
29
30
6
14
13
22
31
30
33
30
27
27
2
I
3
2
5
2
3
4
6
C a lf No.
I* *
2 **
7 ***
.
*Va1ues expressed as percent m igration in h ib itio n .
**Seronegative a t the beginning o f the study.
***C ontrol c a lf .
ND - Not Done
Calves number I and 2 were seronegative fo r BRSV antigen when
the study began.
Seven days po stin fectio n leukocytes from these calves
were in h ib ite d from m igrating in the presence of the v ir a l antigen.
Maximum in h ib itio n values were observed on day 21 when the mean o f in ­
h ib itio n values reached 35%.
Four calves, numbers 3 through 6, were seropositive fo r the
virus p rio r to the experimental BRSV in fe c tio n .
Leukocytes from these
40
Figure 2.
Mean LMI response o f calves follow ing experimental
in fe c tio n with BRSV.
m igration in h ib itio n .
Results expressed as percent
Seropositive calves ( □ ) ,
Seronegative calves ( □ ) , Control c a lf
(O ).
I
I
O
20
30
40
DAYS POSTIffECTION
NOIliaiHNI N O Iim iW I
10
42
calves were in h ib ite d from m igrating a t the beginning of the LMI e v a l­
uation.
Five days a f t e r exposure to the v iru s , leukocytes from these
calves showed a s ig n ific a n t increase (p < 0 .0 2 ) in m igration in h ib itio n .
The maximum in h ib itio n values were observed 21 days postexposure when
the mean o f in h ib itio n values approached 34%.
C alf number 7 was kept
as a noninfected control and it s leukocytes did not show s ig n ific a n t
in h ib itio n in the presence of the v ir a l antigen.
The student's T te s t was employed to analyze the data.
A s ig ­
n ific a n t d ifference (p < 0 .0 1 ) in m igration in h ib itio n existed between
the seronegative and seropositive calves before and a t seven days a f t e r
exposure to the v iru s .
Differences in the migration in h ib itio n of the
two groups a fte r seven days were not s t a t is t ic a lly s ig n ific a n t.
Migra­
tio n in h ib itio n of leukocytes from the s ix infected calves was s ig n if i­
cantly greater (p < 0 .0 0 2 ) than th a t of the control c a lf .
C alf number 2 was reinfected with BRSV 85 days a f t e r the f i r s t
experimental in fe c tio n .
was observed.
No temperature response or c lin ic a l illn e s s
As shown in fig u re .3, m igration in h ib itio n (46%) was
observed seven days a f t e r the re in fe c tio n with BRSV.
Leukocytes from
this c a lf were also tested against the IB R /PI-3 antigen.
In h ib itio n
of leukocyte m igration was observed in w ells containing BRSV antigen
but not in wells containing the IB R /P I-3 antigen.
The c a lf was then
vaccinated by intranasal in s t illa t io n with the IB R /P I-3 Nasal gen.
The
results o f the LMI te s t on leukocytes from this c a lf a f t e r vaccination
-• *
F ig u re
3.
LMI response o f c a lf number 2 a fte r two
experimental in fectio n s with BRSV.
Values
expressed as percent m igration in h ib itio n .
C a lf number 2
( □ ) , Control c a lf ( O ) . .
I
Migratio n I n h ib it io n
I s i Ex p ,
I nfection
Weeks Po stinfectio n
I nfection
45
are shown in ta b le 3.
T a b le
3.
LMI T e s t w i t h
BRSV a n d
IB R /P I-3 A n tig e n s *
Days P ostinfection with IB R /P I-3 Nasalgen
14
7
0:
C alf No** * *
BRSV
IB R /PI-3
2
43
4
BRSV
IB R /PI-3
BRSV
IB R /PI-3
20
31
36
ND**
*Values expressed as percent m igration in h ib itio n .
**ND - Not done.
***T h is c a lf was infected twice previously with the BRSV.
Delayed H yp ersen sitivity Skin Test
Results of the skin tests are shown in table 4.
C alf number 6
developed an increase in skinfold thickness approximately 60 minutes
a fte r the intradermaI in je c tio n of the BRSV antigen.
diminished by 10 hours p o stin je c tio n .
The reaction
A ll of the.calves infected with
BRSV had v is ib le reactions a t 24, 48, and 72 hours a f t e r antigen in je c ­
tio n .
In,most o f the calves maximum skinfold thickness occurred 48
hours a fte r intradermal in je c tio n and were s t i l l present a t 96 hours
p o stin je c tio n .
C a lf number 7 did not show a response to the te s t a n t i­
gen a t 48 or 72 hours but did develop a small increase (1 .0 mm) in
skinfold thickness a t 24 hours p o s tin je c tio n .
The resu lts with the
v iru s -fre e control antigen were uniformly negative and are not pre-
46
sented in the ta b le .
T a b le
4.
D e la y e d H y p e r s e n s i t i v i t y
C alf No.
Skin Reaction
I hr.
S k in T e s t w it h
BRSV A n t i g e n
Skinfold Thickness
48 hr.
I hr.
48 hr.
I
-
+
0.0
4.0
2
-
+
0.0
3.0
3
-
+
0.0
4.0
4
-
+
0.0
5.0
5
-
+
0.0
5.0
6
+
+
3.0
6.0
0.0
0.0
7*
aIncrease in skinfold thickness in m illim e te rs .
*Noninfected control c a lf .
Serological Response
The calves were tested fo r the presence of serum antibody using
the IFAT as previously described.
Table 5 shows th a t the calves in ­
fected with BRSV developed increasing levels of serum antibody s p e c ific
fo r the BRSV.
47
Table 5.
Serological Response to BRSV In fec tio n
C alf No. .
Preinfection
P ostinfection
I
0
+3
2
0
3
3
+1
. +4
4
+1
+4
5
+2
+4
.6
+1
+3
7 (C o n tro l)
+1
+1
Rated s u b jectively from 0 (negative) to a +4 (very in te n s e ).
.
CHAPTER 5
DISCUSSION
Previous studies have assessed the immunological response of
■ y•
c a ttle to BRSV in fe c tio n through the use of serological techniques.
BRSV s p e c ific nasal secretory and serum antibodies are detected f o l ­
lowing both natural and experimental BRSV infections (76, 88, 104).
The present study indicates th a t a cell-m ediated immune response is
also e lic it e d follow ing experimental intranasal BRSV in fe c tio n of
calves.
The LMI te s t under agarose is considered an in v itr o c o rrelate
of CMI (26, 31, 71, 107).
Using the d ire c t technique, whereby antigen
is incubated d ire c tly with the m igrating peripheral leukocytes, presence
of m igration in h ib itio n correlates well with the development
layed cutaneous h y p e rs en s itiv ity reactions (2 ).
of de­
This is interpreted
as o ffe rin g fu rth e r support fo r the use of the LMI te s t as a re lia b le
in v itr o co rrelate of CMI.
In the present study, using BRSV antigen,
a good co rrelatio n was observed between the results o f the LMI te s t
and the development o f delayed h y p e rs en s itiv ity skin reactions.
The s ix calves experim entally infected with BRSV developed a
detectable in v itr o response between fiv e and seven days postinfectidn.
The response, as measured by the LMI te s t using peripheral blood leuko­
cytes, reached a peak between 7 and 21 days a fte r calves were infected
with the v iru s .
The difference in m igration in h ib itio n of the sero­
49
negative calves versus the seropositive calves before and a t seven days
po stinfection could re la te to the la t t e r having had p rio r exposure to
the v iru s .
Presumably they would possess an expanded population of
sensitized lymphocytes s p e c ific fo r BRSV.
This population of sensi­
tiz e d lymphocytes would be capable of mounting a response much e a r lie r
a fte r v ir a l in fe c tio n .
Leukocytes from two of the seropositive calves
did develop maximum in h ib itio n values by seven days po stin fectio n .
The
other calves did not show maximum in h ib itio n values u n til 21 days a f t e r
in fe c tio n with BRSV.
The decrease in in h ib itio n values observed in
a ll of the calves by day 42 may r e fle c t the decreased number of sensi­
tiz e d lymphocytes in the peripheral c irc u la tio n .
One of the calves was reinfected with BRSV 85 days a fte r the
f i r s t experimental in fe c tio n .
As shown in fig u re 3, leukocytes from
th is c a lf developed a more pronounced m igration in h ib itio n (46%) which
was detected by seven days p o stin fe ctio n .
These resu lts suggest a
secondary or anamnestic type of response.
Gadol e t a l . (30) have
demonstrated th a t pulmonary as well as splenic T lymphocytes e x h ib it
memory and are therefo re capable of producing a secondary response.
In
th e ir study, using the macrophage migration in h ib itio n t e s t , they de­
tected a local anamnestic CMI response in the re sp irato ry tr a c t using
both lung wash and splenic T lymphocytes.
The response appeared e a r l­
ie r a ft e r challenge than the primary response but the in te n s ity was
not s ig n ific a n tly g re a ter.
In the present study, the one c a lf re in ­
50
fected with BRSV is not s u ffic ie n t to d e fin ite ly establish th a t a sec­
ondary response was e lic it e d but th is observation does o ffe r evidence
to support the need fo r fu tu re study in th is area.
The mechanism o f in h ib itio n observed in the LMI te s t under
agarose has been investigated and found to be re la ted to a soluble
product o f activated lymphocytes.
This b io lo g ic a lly a c tiv e mediator,
a lymphokine, is designated leukocyte in h ib ito ry fa c to r (L IF ) and has
been shown to s e le c tiv e ly in h ib it the m igration of polymorphonuclear
(PMN) leukocytes (91, 9 2 ).
Both T and B lymphocytes appear to be
capable of producing lymphokines, but the weight o f evidence continues
to suggest th a t lymphokine production is predominantly a T c e ll func­
tio n (5 3 ).
The mechanism responsible fo r the in h ib itio n observed in
th is.stu d y was not d e fin ite ly established.
Microscopic examination
of the m igrating c e lls did in d icate th a t the PMN leukocyte was the
prin cipal c e ll involved in the m igration and were th e .c e lls sensitive
to the in h ib itio n phenomenon.
S p e c ific ity of the LMI response was demonstrated using the
IB R /PI-3 an tig en .
I f the in h ib itio n observed was the re s u lt of a non­
s p e c ific response to foreign antigen, in h ib itio n should also occur when
the IB R /PI-3 antigen was used in the LMI te s t with lymphocytes sensi­
tize d to the BRSV antigen. ' As shown in table 3, no in h ib itio n was
detected in the presence o f the IB R /P I-3 v ir a l antigen p r io r to the
intranasal challenge with the IB R /P I-3 Nasalgen.
51
At the conclusion of the LMI evaluations, the calves were
tested fo r the presence of. delayed, h y p e rs en s itiv ity skin reactions.
The experim entally infected calves developed intradermal reactions
which reached maximal in te n s ity between 48 and 72 hours a fte r the
in je c tio n of antigen.
Although h is to lo g ic a l examination of these
reactions; was not performed, the time frame in which the reactions
developed: in dicate th a t they were delayed h y p e rs e n s itiv ity reactions.
One animal developed an increase in s k in fo ld thickness approximately
60 mintues a fte r the intradermal in je c tio n of BRSV antigen.
The
e a rly appearance of th is reaction suggests th a t i t was mediated by
humoral antib odies, the most probable explanation being a type I I I
or Arthus reaction.
The.animal did develop an indurated
reaction th a t
reached maximal in te n s ity between 48 and 72 hours p o stin je c tio n .
The
control c a lf did not show a response a t 48 or 72 hours but did devel­
op a small increase (1 .0 mm) in s k in fo ld thickness 24 hours a fte r the
antigen in je c tio n .
The c a lf did possess a low t i t e r o f serum antibody
to BRSV as determined by the I FAT.
The increase in skin fo ld thickness
may have been the re s u lt of an Arthus reaction.
The calves were also evaluated fo r a serological response to
the experimental in fe c tio n . Results from the IFAT in d icate th a t the
calves responded with a humoral response and developed increasing t i ;
ters of serum antibody fo r BRSV.
■
The control c a lf did not show an in ­
crease in serum antibody t i t e r fo r BRSV during the te s t.p e rio d .
52
The development of cell-m ediated immunity to BRSV in fectio n
does not necessarily imply th a t th is response contributes s ig n ific a n tly
to the resistance or recovery from
the in fe c tio n .
The mode of virus
re p lic a tio n and c e ll to c e ll spread appear to be important factors in
determining the r e la tiv e importance o f cell-m ediated and humoral re f
sponses in combating v ira l in fectio ns (1 1 5 ). Humoral immunity appears
to be the predominant defense mechanism against those viruses th a t
spread by an e x tra c e llu la r ro u te .
S pecific antibody may coat the v ir a l
■ )'' .'V
p a rtic le s f a c ili t a t i n g th e ir ingestion by phagocytic c e lls or ly s is of
the v ir a l p a rtic le s may occur through the a c tiv a tio n o f the complement
system.
As a r u le , viruses th a t bud from the surface membranes of in ­
fected c e lls or th a t cause the incorporation of v iru s -s p e c ifie d a n ti­
gens in the in fected c e ll's surface membrane are those th a t induce CMI.
BRSV has been shown to bud from the surface of virus-m odified membranes
and to spread from c e ll to c e ll via in te r c e llu la r bridges (60, H O ).
In studying the CMI response to BRSV in fe c tio n i t is necessary
to is o la te i t from as many other factors as possible.
the illu s io n o f separateness may be wrongly im plied.
In doing th is
Both humoral and
cell-m ediated immune responses are components of an overall defense
mechanism and may often overlap one another.
The
recovery or re s is ­
tance to a v ir a l in fe c tio n is probably dependent upon the fin e ly tuned
in te ra c tio n of the various defense mechanisms comprising both s p e c ific
and nonspecific fa c to rs .
53
Secretory antibody appears to function in providing resistance
■ .i
to BRSV in fe c tio n in c a ttle (7 8 ). Recent studies, with human RSV, also
in d ic a te th a t high levels of secretory antibody may provide a curative
e ffe c t and presumably th is could hold true in c a ttle although work in
this area is lacking (6 8 ).
Serum antibody's involvement in defense
against BRSV in fec tio n could occur once pulmonary inflammation devel­
oped.
With th is inflammation, an in flu x of inflammatory c e lls together
with serum antibody might contribute in combating the in fec tio n (5 3 ).
A cel I -mediated
immune response would appear to provide an e ffe c tiv e
mechanism by which in fected c e lls could be lysed before release of in ­
fe c tiv e v iru s .
The CMI response could be mediated by the production
of lymphokines, with the recruitment o f a lv e o la r macrophages and other
c e ll types, or by the d ire c t cytotoxic a c tiv ity of sensitized T lympho­
cytes.
Presumably small numbers of s p e c ific a lly sensitized T lympho­
cytes, upon contact with appropriate antigen, produce s u ffic ie n t quan­
t i t i e s o f lymphokines to e ffe c t changes in the host's defense mechan­
isms (115).
The net re s u lt of th is a n tig en -sp ec ific lymphokine pro­
duction is the recruitm ent of nonsensi t i zed lymphocytes, macrophages, ■
and neutrophils.
This recruitm ent o f nonsensi t i zed lymphocytes may
act as an am p lific atio n system whereby these lymphocytes are induced
to produce lymphokines th a t augment the i n i t i a l l y small response to
the v ir a l in fe c tio n (5 3 ).
54
A lveolar macrophages co n s titu te the f i r s t lin e o f defense in
the lung.
In addition to a crucial ro le in nonspecific c e llu la r de­
fense they also comprise key e ffe c to r c e lls in CMI.
Several of the
■
lymphokines act s p e c ific a lly upon macrophages, re s u ltin g in"the re ­
cruitm ent, a c tiv a tio n , and trapping of these phagocytes a t the reac­
tio n s it e ,
Macrophages, a ttra c te d to the s it e , appear capable of
preventing v ir a l spread by discouraging the formation o f in t e r c e ll­
u lar bridges (9 3 ).
This response would appear to co n s titu te a major
mechanism fo r the defense o f the bovine lung in lim itin g BRSV in ­
fe c tio n .
CHAPTER 6
SUMMARY
Results of the LMI te s t in d ic a te th a t a CMI response was
e lic ite d follow ing the intranasal in fe c tio n with BRSVs s tra in VC494.
The response, as measured in migration in h ib itio n , was detected
as e a rly as fiv e days p o stin fe ctio n .
The mean o f in h ib itio n values .
reached a maximum of 34%, 21 days a f t e r the calves were infected
whereas the corresponding value before in fe c tio n was 8.7%.
An enhanced
in h ib itio n which developed more ra p id ly was observed with leukocytes
from one c a lf rein fected with BRSV.
This observation was suggestive
■/
of a secondary or anamnestic CMI response.
Delayed h y p e rs en s itiv ity skin tests were performed a fte r the
LMI evaluations were concluded.
The s ix experim entalIy infected calves
developed intradermal reactions which reached maximal in te n s ity be­
tween 48 and 72 hours po stin jectio n of the BRSV antigen, whereas the
control c a lf gave negative re s u lts .
Results of the IFAT showed th a t,
serum antibody levels also increased in the calves follow ing in fe c tio n .
Results of th is study indicated th a t both humoral and c e llmediated immune responses were e lic it e d follow ing experimental, in fe c ­
tio n of calves with the BRSV, s tra in VC-494.
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