ELSEVIER Veterinary Immunology and lmmunopathology

50 (1996) 79-91

Veterinary immunology

and immunopathology

Cell-mediated immune responses of lambs to experimental infection with bovine respiratory

syncytial virus

A.K. Sharma, Z. Woldehiwet *

1Jniorrsity oj’Liurrpoo1. Depurtmmt r,f‘Veterintrry Pathology. Vc,terirury Field Station. Leuhurst, .Nccton.

Wirrul. Lti4 7TE. UK

Accepted I June 1995

Abstract

_

Primary infection of lambs with bovine respiratory syncytial virus (RSV) was characterised by the presence of virus-specific cytotoxic cells in the peripheral blood as early as 5 days post-infection. These effector cells lysed virus-infected autologous targets in a self-restricted manner. Depletion techniques revealed that cytotoxic activity was largely due to OvCDS + cells. Neutrophils obtained from experimentally infected lambs 5 to IO days post-inoculation exhibited significant cytotoxic activity in the presence of bovine RSV-specific antiserum. During the same period there was a significant lymphoproliferative response to live or inactivated bovine RSV. Lymphoproliferative and cytotoxic activity coincided with the clearance of the virus from nasal secretions.

1. Introduction

Bovine respiratory syncytial virus (RSV) has a narrow host range and, unlike human RSV, does not infect inbred mice. However, lambs are increasingly being used as experimental models to study the pathogenesis and immune responses to bovine RSV (Al-Darraji et al., 1982; Sharma and Woldehiwet, 1990; Meehan et al., 1994) and recent

reports suggest that they could be used to investigate human RSV too (Lapin et al.. 1993).

* Corresponding author

0165.2427/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDf 0165-2427(95)05488-X

80 A.K. Shurmu, 2. Woldehiwcr/ Vererinury Immunology and Immunoputholo~y 50 (1996) 79-91

In calves, infections with bovine RSV frequently occur in the presence of high levels

of circulating antibodies (Stott and Taylor, 1985). In humans too there is a poor correlation between neutralising antibody levels and resistance to RSV infection (La Via

et al., 1992). Furthermore, immunoglobulin (Ig)-deficient children are not particularly

more susceptible to infection with RSV than immunocompetent children. but children with defective cellular immunity suffer from prolonged RSV infection (Fishaut et al.,

1979). The role of cell-mediated immune responses in recovery from respiratory disease due

to bovine RSV is not yet clearly defined but it has recently been shown that depletion of

T cells rendered calves more susceptible to experimental infection with bovine RSV

(Taylor et al., 1993), and some cytotoxic activity is present in the peripheral blood of experimentally infected lambs (Sharma and Woldehiwet, 1991).

In the present study we report the presence of virus-specific and self-restricted

cytotoxic activity in the peripheral blood of lambs experimentally infected with bovine

RSV.

2. Materials and methods

2.1. Experimental infection

2.1.1. Lambs Fourteen conventionally-reared male cross-bred lambs, aged between 10 and 14

weeks were used. Secondary lamb testis cell (LTC) cultures were prepared from all lambs for subsequent use as autologous or allogenic target cells in cytotoxicity assays.

Eight lambs (Group I> were inoculated with tissue culture fluid containing 6.3 log10 TCIDSO per ml of bovine RSV by the combined respiratory route as described earlier

(Sharma and Woldehiwet, 1990). Six control lambs (Group 2) were inoculated with the same volume of uninfected cell culture fluid.

2.1.2. Viruses A strain of bovine RSV (BRSV 66) passaged three times in LTC, was maintained and

used as described earlier (Sharma and Woldehiwet, 1990). A non-cytopathic strain of Border disease virus (ncpBDV L991) maintained in LTC was used to prepare control target cells (Woldehiwet and Hussin, 1994).

2.1.3. Monoclonal antibodies Monoclonal antibodies which recognise the OvCD4 + (helper) and OvCD8 + (cyto-

toxic/suppressor) T cells were used for the selective depletion of lymphocytes as described earlier (Woldehiwet and Hussin, 1994).

2.1.4. Collection of samples Heparinized peripheral blood samples collected on three occasions before immunisa-

tion (8, 4 and 0 days before inoculation and on days 5, 10, 15 and 21 post inoculation (pi) were used in cytotoxicity or lympho-proliferation assays. Serum samples were

A.K. Sharma, Z. Woldehiwet/ Veterinary Immunology und Immunoputholo~y SO (1996) 79-91 8 I

collected on the day of inoculation (Day 0) and on Days 4, 8, 12, 16, 21, 28. 35, 42, 49 and 56 pi. Lambs were observed daily for clinical signs such as coughing and nasal

discharges. Rectal temperatures were recorded and nasal swabs collected daily up to 14 days post inoculation.

2.1.5. Virus isolation Nasal swabs were processed for virus isolation on LTC cultures as described earlier

(Sharma and Woldehiwet, 1990). Isolates were confirmed as bovine RSV by indirect immunofluorescence using monoclonal antibodies against the F and N proteins of

bovine RSV, kindly donated by J.A. Kramp, Central Veterinary Institute, Lelystad, The

Netherlands.

2.1.6. Virus neutralising (VN) antibody assay Micro-neutralisation tests were performed on LTC cultures, in 96-well plates. Titra-

tions were carried out using constant virus and variable serum as described earlier (Sharma and Woldehiwet, 1992).

2.2. Cytotoxicity assay

2.2.1. Effector cells Peripheral blood mononuclear cells (PBMC) and neutrophils were used as effector

cells in cytotoxicity assays. Mononuclear cells were separated from heparinized blood

with Ficoll-Paque gradient centrifugation as described earlier (Woldehiwet and Hussin. 1994). The remaining cell pellet was used as a source of neutrophils after lysis of red cells by the method of Carlson and Kaneko (1973). The neutrophils were used as effector cells for the antibody-dependent neutrophil cytotoxicity (ADNCl assays.

PBMC were used as effector cells before and after selective depletion of the OvCD4 + or OvCD8 + cell fractions as described earlier (Woldehiwet and Hussin, 1994). The effectiveness of depletion was verified by flow cytometry. PBMC and neutrophils were thoroughly washed, assessed for viability and finally suspended in RPM1 1640 medium to give the desired effector: target cell (E: T) ratios.

2.2.2. Target cells A total of four types of target cells were used in cytotoxicity assays: autologous

bovine RSV-infected LTC, autologous BDV-infected LTC, autologous uninfected LTC and allogenic bovine RSV-infected LTC. The LTC were inoculated at a multiplicity of infection (moi) of three or sham-exposed for 2 h and incubated for 16 h at 37°C in Eagles MEM, without foetal calf serum (FCS). At the end of incubation, the monolayers were trypsinised, washed and viable cells counted. In total, 10 X lo6 viable cells were labelled with 100,Ci of Na,“CrO, (Amersham) for 2 h at 37°C. Labelled cells were washed twice, incubated for 1 h and washed again before final suspension in RPM1 1640 to give a concentration of 4 X lo4 cells ml-‘.

2.2.3. Antisera Heat-inactivated polyclonal ovine antiserum against bovine RSV was used for

antibody-dependent neutrophil cytotoxicity (ADNC) assays using optimum dilutions of serum (1 : 200).

82 A.K. Shormu. Z. Woldehiwrt/ Vetcrinury Immunology und Immunoppotholo,qy 50 (1996) 79-91

2.2.4. Chromium release assay

The chromium release assay was carried out as described by Woldehiwet and Hussin (1994). Assays were carried out in triplicates in LP3 tubes (Sarstedt, Numbretch) at E : T ratiosof50:l and25:l.

Briefly, 250,l of labelled target cells were mixed with 250,l of effector cells and the tubes were centrifuged at 150 g for 2 min to enhance contact between effector and

target cells. The tubes were then incubated at 37°C for 5 h in an atmosphere of 5%

carbon dioxide. Spontaneous release was determined by incubating target cells with

250,l RPM1 1640. Total release was determined by treating the target cells with 2.5%

Triton X-100 (Sigma Chemical Company Ltd., Poole, Dorset) at the end of 5 h and

incubating the tubes further at 45°C for 30 min in order to ensure the maximum release of bound chromium. A total volume of lOO,l of supematant as collected in duplicate

tubes and the radioactivity counted in a gamma counter (1270 Rack Gamma II, LKB, Wallac, Tukru).

For assaying antibody-dependent neutrophil cytotoxicity (ADNC) or antibody-inde-

pendent neutrophil cytotoxicity (AINC), 200,l autologous target cells were incubated

for 30 min with 50,l of optimally diluted antiserum or medium followed by addition of 250,l of effector neutrophils. The cytotoxicities of different fractions of effector cells

were expressed as % specific lysis which was calculated as follows:

% specific lysis = cpm” Experimental-cpm Spontaneous

cpm Total-cpm Spontaneous x 100 (1)

* = counts per minute

2.3. Lymphocyte proliferation (LP) assay

2.3.1. Preparation of purified and inactivated virus

Purified virus was prepared from virus-infected LTC culture fluids, as described by Trepanier et al. (1981). Briefly, LTC cultures were infected with bovine RSV at a multiplicity of infection (moi> of 0.2 and harvested by three cycles of freezing and thawing, when about 80% of the monolayer showed CPE. The infected culture fluid was cleared of the cell debris by centrifugation at 3000 g for 30 min at 4°C. The clarified supernatant was precipitated overnight with 10% (w/v) polyethylene glycol 6000 at 4°C. The precipitate was collected by centrifugation at 10000 g for 30 min at 4°C in a Sorvall T865 fixed angle rotor (Sorvall Combi Ultracentrifuge, Du Pant) and resus- pended in a volume of cold TNE (0.05M Tris, O.OlM NaCl and 0.001 M EDTA, pH 7.8) buffer equal to one hundredth of the initial volume.

The concentrated virus preparation was further purified on a discontinuous 30/60% sucrose density gradient and centrifuged at 100000 s at 4°C for 18 h in a swing out rotor (Sorvall AH 629, Du Pont UK Ltd. Stevenage, UK). Virus containing fraction was pelleted at 100000 g for 2 h in TNE buffer.

Purified virus preparations were inactivated with beta-propiolactone (Sigma Chemical Company Ltd., Poole, Dorset) at a dilution of 1 : 4000 for 18 h at 4°C and then incubated

at 37°C for 2 h to hydrolyse the residual beta-propiolactone (Koprowski. 1973).

Inactivation was demonstrated by the failure of the viral suspension to infect LTC. Live and inactivated virus preparations were aliquoted and stored at -70°C until

required. Throughout the study, the same batch of virus and antigen was used. Control

antigens were similarly prepared from uninfected LTC.

2.3.2. Lvmpho-proliferatiue assay The iympho-proliferative (LP) assays were performed as described by Hussin and

Woldehiwet (1994). Briefly, PBMC were suspended at a concentration of 1 X 10h cells

per ml in RPM1 1640 medium containin g L-glutamine (2mM), penicillin (100 i.u per ml), streptomycin (100,g per ml), HEPES (20 mM), 2-mercaptoethanol (0.5 mM) and

5% autologous serum. A volume of 300~1 of PBMC suspension as dispensed into

triplicate wells of U-bottomed microtitration plates containing 25,l of optimum concen- tration of live or inactivated virus (equivalent to an moi of 5) or 25,l of the control antigen. Plates were incubated at 37°C in an atmosphere of 58 carbon dioxide for 72 h.

After incubation, the cultures were pulsed overnight with 1,Ci of [“HI thymidine (Amersham International Plc, Amersham) in 25,l per well and subsequently harvested

onto filtermats (Skarton, A.S. Herbyen) by a multiple sample harvester (Skarton). The radioactivity in filtermats was determined by a scintillation counter (Packard Liquid Scintillation Analyzer, TRI CARB 1900 TR) and expressed as disintegration per minute

(dpm).

2.3.3. Analysis of data Low levels of [3H] thymidine uptake always occurred in unstimulated cultures.

Specific responses were, therefore, calculated as the difference (D) between dpm of stimulated and unstimulated cultures (Schultz. 1982). All values were first converted into their square roots before calculating D values (Woldehiwet, 1987). Values of samples obtained from infected lambs were compared with those obtained from control lambs by Student’s r-test. The paired t-test was used to compare sequential changes within groups.

3. Results

All lambs were healthy and did not show any clinical signs before experimental inoculation. After inoculation, infected lambs showed mild clinical signs characterised by nasal discharge, coughing and mild febrile reaction on days 3 and 4 pi. Control lambs remained free of clinical signs throughout the period of experiment.

3.1. Virus isolation

Bovine RSV was isolated from all eight lambs in Group 1, from day 1 pi through to day 4 pi and from seven lambs on day 5 pi, five lambs on day 6 pi, three lambs on day 7 pi, and one lamb on day 8 pi. The lambs in Group 2 remained negative for bovine RSV on all days of sampling.

84 A.K. Sharmu. 2. Woldehiwet/ Veterinury Immunology and Immunoprrthology 50 (1996) 79-91

3.2. Virus neutralising antibody response

In lambs of Group 1, significant (P < 0.001) VN antibodies were first detected on day 8 pi (4.9 + 0.6; log2, mean + sd). Peak VN titres of 8.0 f 0.1 were recorded 21

days pi, gradually declining thereafter. No bovine RSV-specific VN antibodies were detected in serum samples obtained from control lambs.

3.3. Cytotoxicity in PBMC

Spontaneous release of radioactive chromium from target cells ranged between 10

and 22%. There was no significant specific release when PBMC from bovine RSV-in- fected lambs were incubated with uninfected autologous or allogenic targets at E : T ratio

of 50 : 1 or 25 : 1. Significant bovine RSV-specific cytotoxicity was first detected 5 days after infection (Table 1). Peak levels of cytotoxicity (28.05 k 7.89%) were recorded 10 days pi. When an E: T ratio of 50: 1 was used, significant cytotoxicity continued to be

detected up to 21 days pi, the last day of sampling (Table 1). Significant but lower levels of cytotoxicity were recorded up to 15 days pi when an E : T ratio of 25 : 1 was used (Table 1). There was no bovine RSV-specific cytotoxicity in effector PBMC obtained

from control lambs irrespective of the E: T ratio used (Table 1).

3.4. Virus-specificity and ‘self-restriction’

Effector PBMC obtained from infected lambs preferentially lysed bovine RSV-in-

fected autologous LTC, compared with the uninfected autologous LTC. BDV-infected autologous LTC or bovine RSV-infected allogenic LTC on all sampling days. For example, on day 10 pi, there was a significant specific release of 28.05 + 7.89% when autologous bovine RSV-infected targets were used compared with no significant specific release when BDV-infected autologous targets, uninfected autologous targets or bovine

Table I Cytotoxic activity in the PBMC obtained from infected and control lambs against bovine RSV-infected

autologous targets (per cent, mean + sd)

day pi

Lambs

0

5

IO

I5

21

Group I Group 2

Group 1 Group 2

Group I Group 2

Group I Group 2

Group I

Group 2

Cytotoxicity at E: T ratio of

50: I 25: I

4.37 * I .82 3.88 * I .03 4.52+ 1.66

I I .57 k 3.38

3.15k 1.62

28.05 & 7.89

4.42 i 0.87

14.1 k4.17

5.32 + I .67

8.34k 1.5 . 4.63 t I

4.6 I k 0.63

. . 8.73 f 2.74 - 1 ’ 3.57 + 2.44

s I 20.56k7.1 I . ‘ _

4.47 + I .22

I 1 8.58 f 5.22 1 3.42 k 2.54

6.57 f I.37

5.27& I.15

*. P <O.Ol; **‘P<o.ool

A.K. Sharmu. 2. Woldehiwvt/ Veterincrry Immunology und Immunoputholo~y 50 (1996) 79-91 85

Table 2

Cytotoxic activity in the PBMC against different target cells. on day IO pi, at E: T ratio of 50. I (mean k sd.

per cent specific lysis)

Target cells Cytotoxicities with effector cells obtained from lambs of

Group I (n = 8) Group 2 (n = 6)

Bovine RSV-infected,

autologous LTC

BDV-infected,

autologous LTC

Uninfected,

autologous LTC

Bovine RSV-infected,

allogenic LTC

28.05 5 7.89 * * * 4.42 k 0.87

4.87 + 0.92 4.71 f 1.76

4.66+ 1.91 5.21 * 2.13

5.48 + 0.95 4.67 k 2.39

RSV-infected allogenic targets were used (Table 2). There was no significant cytotoxic-

ity by control PBMC against any of the targets used.

3.5. Effect of selective depletion on cytotoxicity

Two cycles of depletion with specific monoclonal antibodies consistently removed

more than 97% of the desired cell population (Woldehiwet and Hussin, 1994). Depletion of OvCDS + cells abrogated cytotoxic activity of the PBMC obtained from experimen-

tally infected lambs, except on day 10 pi (Fig. I>. On the other hand, significant cytotoxic activities were detected in OvCD4-depleted effector PBMC on days 5. IO and 15 pi (Table 3).

40 -

2 2 - PBMC (undepleted)

E” 30

-

i -=~‘W.~ OvCD4depleted PBMC

.-....9= OvCDSdepleted PBMC

Days post inoculation

Fig. I Effect of depletion of OvCD4 + or OvCDR + cells on cytotoxicity.

86 A.K. Shurmu, Z. Woldehiwet/ Veterinary lmmunolo~y und lmmunoputholc~~~y 50 (1996) 79-91

Table 3

Effect of selective depletion of OvCD4+ or O&D8 + cells on bovine RSV-specific cytotoxicity (E: T = 50: I )

Dpi Group Cytotoxic activities with effector cells obtained from experimentally-infected lambs

Undepleted OvCD4depleted OvCDS-depleted

PBMC PBMC (n = 6) PBMC (n = 6)

0 I 4.37 f I .82 4.64+ 1.24 5.06 + 0.98

2 4.52+ 1.66 4.76 i 0.58 5.62 i 0.94

5 1 I I .57 k 3.38 7.99+3.1 _ 4.85 ri- I 2 3.48 f I .62 4.49+2.51 4.68 + I. I5

IO I 28.05 k 7.89 21.3 +3.31 “ I2 f 2.3 . 2 4.42 f 0.87 4.92 k I .89 3.5 I * 2.5 I

15 I 14.1 k4.17 10.88 + 2.4 - 5.46 * 2. I 2 5.32 f I .67 3.86+ 2.65 4.8 I _+ 2.59

21 I 8.34i_ I .5 6.14k2.22 5.08 f I 2 4.63 i 0.99 4.71 ri_2.19 4.39 I_ I .99

* P<O.Ol; *‘P <O.OOl

3.6. Neutrophil cytotoxicity

Neutrophils obtained from experimentally infected lambs, exhibited significant cyto- toxic activity in the presence of bovine RSV-specific antiserum (ADNC) in samples obtained 5 and 10 days pi, at an E: T ratio of 50: 1 (Fig. 2). No significant antibody-in-

dependent neutrophil cytotoxicity (AINC) was recorded in the neutrophils obtained from experimentally infected lambs on any day of sampling (Table 4). There was no significant AINC or antibody-dependent neutrophil cytotoxicity (ADNC) in the neu- trophils obtained from control lambs.

3.7. Relation between virus shedding, VN antibody and cytotoxic responses

Bovine RSV was cleared from the nasal secretions of all the experimentally-infected lambs when peak levels of virus specific cytotoxic activity were detected in the PBMC

PBMC from infected lambs

PBMC from control lambs

Ncutrophlls from mfected lambs + anteerum

Neulwphils from control lambs + anhsrrum

+ . I I I . I

5 10 15 20 2.5 Days post inocutahon

Fig. 2. Cytoxicity of PBMC and neutrophils of infected and control lambs.

Table 4

Neutrophil cytotoxicity in experimentally-infected and control lambs

Day pi

PMN from lambs in Group I PMNs from lambs in Group 2

+ antiserum no antiserum + antiserum no antiserum

(ADNC) (AINC) (ADNC) (AINC)

0 4.92 k I .46 5.93 i I.87 5.26 +0.97 s.49+ 1.40

5 12.66+_3.81 * - 5.76 + I .43 4.91 kO.77 4.76i 1.61

IO 8.14*2.19 ’ 5.92 f 1.84 4.51* I .04 4.4 * 1.19

IS 5.68 f I .54 5.39 + 3.54 5.05 + I.82 4.43 * 2.5x

21 .5.X6_+ 1.86 5.64 & 2.59 5.23 I I .4s 3.88 f I .74

’ == P < 0.0: * + = P < 0.001

(day 10 pi). Peak VN antibody tines in infected group of lambs were recorded on day 2 I pi, 11 days after virus had been cleared from the nasal secretions (Fig. 3). Therefore, the

elimination of virus from nasal secretions appeared to be related to the cytotoxic activity in PBMC rather than the antibody response (Fig. 3).

3.8. LP responses to inactivated or live bovine RSV

Significant LP responses to inactivated or live bovine RSV were detected in the PBMC of infected lambs 5 to 10 days pi (Table 5). These responses increased progressively, and peak levels of LP responses were recorded on day 21 pi, the last day

of sampling (Table 5). A comparison of the LP responses generated by live virus with those generated by inactivated virus revealed that inactivated virus stimulated signifi- cantly higher LP responses in PBMC obtained from primed lambs on days 5 to 21 pi

“J 1 I I I 1 I 0 5 10 15 20 25 300

Days post lnoc”lahon

Fig. 3. Bovine RSV-specific cytotoxicity in PBL and virus neutralising antibodies in experimentally infected

lambs.

88 A.K. Shurmu. Z. Woldehiwr/ Vrterincrry Immunology and Immunoputholo~y 50 (1996) 79-91

Table 5

Lympho-proliferative responses of the PBMC of infected (Group I) and control (Group 2) lambs to live and

inactivated bovine RSV (mean _+ sd, D values)

DF Lympho-proliferative responses to

Inactivated bovine RSV

Group 1 Group 2

Live bovine RSV

Group I Group 2

0 1.24+4.1 1.5 +5.21 2.08 * 4.93 I .89 * 2.82

5 16.84k4.65 * 0.29 + 3.98 5.48 i 2.89 4.96 f 2.36

IO 23.57 f 3.58 * 0.49 f 4.74 15.74*3.19 * 3.87 * 4.75

I5 36.56k 8.38 * 0.75 * 5.48 32.48k5.14 ’ 2.2 & 2.75

21 62.14k6.45 . -0.6 F4.77 45.37 _t 4.04 * 2.35 & 3.34

’ P < 0.01; DP = days post-inoculation

(P < 0.001; Table 5). PBMC obtained from control lambs did not have significant LP

responses to inactivated or live virus on any day (P > 0.05; Table 5).

4. Discussion

The present study documents the presence of bovine RSV-specific cytotoxic T cells in the peripheral blood of experimentally infected lambs. These effector cells lysed virus-infected autologous target cells in a ‘self-restricted’ manner. This is in agreement with the findings of other workers with human RSV in BALB/c mice (Bangham et al., 1985) and in human adults (Chiba et al., 1989; Issacs et al., 1987). Earlier studies in this laboratory had demonstrated bovine RSV-specific cytotoxic activity in PBMC of experimentally infected lambs (Sharma and Woldehiwet. 1991) but it could not be ascertained in that study whether the cytotoxicity was ‘self-restricted’ or not.

In domestic animals, CTL activity has been shown against Theileria parua (Eugui and Emery, 1981) and Bovid herpes virus (BHVl-1 (Splitter et al. 1988) in cattle, against Equid herpes virus type I (Bridges and Edington. 1987) in horses, against vaccinia virus (Pearson et al., 1981) and Border disease virus (Woldehiwet and Hussin, 1994) in sheep, against canine distemper virus (Shek et al.. 1980) in dogs, and against African swine fever virus (Martins et al., 1988) in pigs.

OvCD8 + cells are the ovine cytotoxic T cell equivalents of mice or humans and their activity is MHC-restricted (Ezaki et al., 1987). In the present study depletion techniques were used to demonstrate that cytotoxic activity was largely due to OvCD8 + cells. However, significant cytotoxicity was also observed with OvCD8-depleted effec- tor PBMC on day 10 pi (P < 0.001) which suggests that OvCD8 + cells may not be the exclusive cell population mediating cytotoxic responses. The possible contribution of OvCD4 + cells, NK cells and lymphokine activated killer (LAK) cells to the overall cytotoxic activity cannot be ruled out. However, induction of enhanced NK activity in a 5 h assay is less likely to occur (Ortaldo and Herberman, 1984). Moreover, ‘self-re- stricted’ cytotoxicity is typical of cytotoxic T lymphocytes and has not been described for either ADCC or NK activity (Ortaldo and Herberman. 1984; Li and Splitter, 1994).

Although classical MHC class I-restricted CTL are considered to be the main

mediators of cytotoxic responses to many viral infections, it has been demonstrated that MHC class II-restricted CTLs also participate in the overall immune response to viruses

(Jacobson and Biddison, 1986). For example, natural infection with human RSV induces

MHC class I-restricted CTL (Bangham et al., 198.5; Munoz et al., 1991), while infection with influenza virus elicits both classes of CTL (Jacobson and Biddison, 1986). The

precise ratio of the class I- and class II-restricted CTLs that mediate immunoprotection appears to vary with different viruses (Jacobson and Biddison. 1986).

It has been suggested that neutrophils may play an important role during early phases

of RSV infections, by acting against RSV infection at the site of viral replication, by

attachment to RSV-infected cells and subsequent release of mediators (Fadan et al.. 1984). Complement-dependent neutrophil cytotoxicity (CDNC) is thought to be another

important defence mechanism against human RSV (Kaul et al., 1984) and other viral

infections (Campos et al., 1982). Faden et al. (1984) reported that adherence of neutrophils to RSV-infected cells is directly proportional to the extent of RSV replica-

tion. Furthermore, RSV-specific antibodies have been shown to enhance the adherence

of PMN to human RSV-infected cells in a dose-dependent manner in vitro. In the present study, significant ADNC but not AINC, was detected at 5 and 10 pi.

Neutrophils from bovine RSV-infected lambs had significant cytotoxic activity against bovine RSV-infected target cells in the presence, but not in the absence, of bovine RSV-specific antibodies. Compared with the cytotoxic T cell activity of PBMC, the

levels of ADNC were significantly lower. However, closely spaced samplings. specially during the first few days of infection could have revealed more information on the role of PMN cytotoxicity. Neutrophils are believed to be the most potent mediators of

antibody-dependent cellular cytotoxicity against RSV in humans (Kaul et al., 1984) and cattle (Grewal et al., 1977). Recently Bradford et al. (1992) reported that both ADNC and AINC were important mechanisms against para-influenza virus type 3 (PIV3)-in- fected targets in vitro.

Lymphocyte proliferation driven by specific antigens or mitogens has been used as a means to define cell-mediated immunity. Several groups of workers have shown that antigen-specific LP responses to human RSV appear in the peripheral blood of naturally infected humans (Scott et al., 1984). However, the responses in many cases are delayed and are not detectable during acute stages of infection. Experimental infection with bovine RSV induced significant LP responses to viral antigens in four of six gnotobiotic calves, and six of 21 conventionally-reared calves (Taylor et al., 1987).

In the present study, LP responses to live and inactivated bovine RSV were detected in the PBMC of infected groups of lambs. Antigen-induced blastogenesis is largely a T

cell-dependent function (Leguit et al.. 1973) and therefore the generation of LP responses in experimentally infected lambs to bovine RSV antigen appears to be due to

antigen-specific T cell activation. Inactivated bovine RSV induced significantly higher LP responses than live virus.

The lower responses to live virus could be due to the possible inhibitory effects of the infectious virus itself (Salkind et al., 1991). In another study (Sharma, 1994) it was observed that infectious bovine RSV significantly inhibited antigen-specific LP re- sponses to Border disease virus.

90 A.K. Sharmu. Z. Woldehiwet/ Vetrrinury Immunology und Immunoputhology 50 f 1996) 79-91

Acknowledgements

The authors thank M.I. Brandon, University of Melbourne, for the donation of monoclonal antibodies against lymphocyte subsets and J. Kramp, The Central Veterinary

Institute, Lelystad, for the donation of monoclonal antibodies against bovine RSV. A.K. Sharma was supported by a grant from the Association of Commonwealth Universities,

London.

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