Reinfection of lambs with bovine respiratory syncytial virus

Research in Veterinary Science, 1992, 52, 72-77

Reinfection of lambs with bovine respiratory syncytial virus R. SHARMA*, Z. W O L D E H I W E T ~ , University of Liverpool, Department of Veterinary

Pathology, Veterinary Field Station, Leahurst, Neston, Wirral, L64 7TE

Eight lambs which were experimentally infected with bovine respiratory syncytial virus (asv) when they were six to eight weeks old were challenged with the same virus seven months later. After reinfection, lambs developed mild clinical disease and the virus was isolated from nasal swabs from three lambs and peripheral blood from two lambs. Reinfection resulted in changes in peripheral blood cell populations. There was an early increase in the number of CD8+ T lymphocytes and B (LCA p220+) lymphocytes but the proportions of CD4+ and CD4-CD8- T lymphocytes were significantly reduced. Peripheral blood mononuclear cells obtained from lambs reinfected with bovine asv showed significantly higher responses to bovine RSV antigen in vitro than those obtained from control lambs but their responses to the mitogen phytohaemagglutinin were significantly lower than in control lambs. RSV-specific IgG, IgM and IgA levels of serum samples obtained 10 days after challenge were significantly higher than those of serum samples obtained before challenge. E P I D E M I O L O G I C A L studies indicate that human adults with pre-existing serum antibodies to respiratory syncytial virus (RSV) can be reinfected experimentally (Mills et al 1971). Stott et al (1984) found that calves were susceptible to bovine RSV as early as three weeks after a primary infection. The factors which contribute to the occurrence of RSV reinfection are not known. After analysing several factors which might contribute to the recurrence of RSV infection in humans, Beem (1967) concluded that reinfection was not due to antigenic variation, age of patient at the time of first infection or length of time between primary and secondary exposure. *Present address: Department of Veterinary Microbiology, College of Veterinary Science, Haryana Agricultural University, Hissar 125400, India "~Reprint requests to Z. Woldehiwet

Lambs experimentally infected with RSV develop mild respiratory disease (Sharma and Woldehiwet 1990a) and primary infections are accompanied by alterations in lymphocyte subpopulations in peripheral blood (Sharma et al 1990) which may account for the increased susceptibility to bacterial superinfection (Sharma and Woldehiwet 1990b). The purpose of the present study was to examine whether experimentally infected lambs were susceptible to reinfection.

Materials and methods

Bovine respiratory syncytial virus A strain of bovine RSV (BRSV 66), passaged in lamb testis cells three times, was maintained and used as previously described (Sharma and Woldehiwet 1990a).

Lambs Sixteen conventionally reared Suffolk crossbred lambs were used. The first group of eight lambs was infected with bovine RSV when the lambs were six to eight weeks old (Sharma and Woldehiwet 1990a). The second group of eight lambs was inoculated with tissue culture fluid and kept in a separate house. When the lambs were nine to 10 months old, the infected group was challenged with 20 ml of bovine Rsv-infected tissue culture fluid. Lambs in group 2 were inoculated with sterile cell culture fluid. Both groups were kept under clinical observation for nine days as described by Sharma and Woldehiwet (1990a).

Virological studies Nasal swabs collected before challenge and three, five, seven and 10 days after challenge 72

Reinfection of lambs with bovine respiratory syncytial virus were processed for virus isolation as previously described (Sharma and Woldehiwet 1990a).

73

rinsed 10 times with demineralised water containing 0-025 per cent Tween 20, 0.025 per cent Tween 80 and 0.87 per cent sodium chloride (ELISAwash).

Collection of blood samples Heparinised blood samples collected on days 0, 5, 10 and 14 after challenge were used to establish the distribution of lymphocyte subpopulations and for the lymphocyte transformation tests. Mononuclear cells were separated from diluted blood on a Ficoll-Paque column (Sharma et al 1990). Serum samples collected on days 0, 10, 14 and 21 after challenge were used to establish class-specific antibody responses by ELISA.

Indirect double antibody sandwich assay

MicrO-ELISA polyvinyl plates (PVC, E1A plates, Flow Laboratories) were coated with 50 gl of optimally diluted anti-BRSVmAb, specific to the F and N proteins, kindly provided by Dr F. Westenbrink, Central Veterinary Institute, Lelystad, Utrecht, The Netherlands. After overnight incubation at 4°C, mAb was poured offand the remaining free binding sites on the wells were blocked by 100 gl of 0.2 per cent bovine serum albumin (BSAFraction V, Sigma) Identification of lymphocyte subpopulations in sample diluent. After one hour's incubation at The number of lymphocyte subpopulations 37°C, the BSAsolution was poured off and the plates were rinsed in ELISAwash. BRSVantigen preparations was established using monoclonal antibodies by (50 gl per well) and appropriate controls were then flow cytometry as described previously (Sharma dispensed into antibody-coated wells and the plates et al 1990). incubated for a further one hour at 37°C. Fourfold dilutions of the test sera and negative and positive Lymphocyte transformation assays controls were added in duplicate and the plates Lymphocyte transformation responses of incubated for one hour at 37°C. Fifty gl of optimally diluted peroxidase-conmononuclear cells to phytohaemagglutinin (PHA) jugated antibodies against sheep IgG or IgM were and bovine RSV were assayed as described earlier then added to each well, except substrate controls, (Sharma and Woldehiwet 1990c). and the plates were incubated for one hour at 37°C. Finally, 100 gl of freshly prepared substrate Assay of humoral immune responses solution were added to each well and the plates BRSV-specificIgG and IgM antibodies in serum were left in the dark at room temperature until samples were titrated in an indirect double anti- the development ofcolour in positive serum sambody sandwich assay (IDAS)using anti-BRSVmon- ples. The reaction was stopped by the addition oclonal antibody (mAb), as coating antibody, of 50 gl of 4N sulphuric acid per well. The absorband peroxidase conjugated IgG fraction of swine ance of each well was determined by a micro anti-sheep IgG or swine anti-sheep IgM (Eivai plate reader (MR 700, Dynatech) using a test waveBios Lab, West Sussex). Virus-specific IgA anti- length of 490 nm, calibration setting of 1.00 and bodies were detected by the antibody capture a threshold of 1.99. The machine was blanked assay (ACA)method, using IgG fraction of swine with substrate controls. anti-sheep IgA (a-chain specific) (Eivai Bios Lab), The serum titre was taken as the dilution scoras capture antibody, and anti-BRSV mAb. The ing one matrix unit above the value obtained ELISAtest was performed as described by Kimman with the standard negative serum in BRSVantigenet al (1987b) with some modifications, using 0.05 coated wells in the same plate as described by M carbonate-bicarbonate (pH 9.6) as coating Kimman et al (1987b). The matrix for each plate buffer, 0-5 M tris buffer (pH 7.4) containing 1 was set according to the maximum E 490 value mM EDTA, 1 M sodium chloride, 0.1 per cent obtained with the standard positive serum. One bovine serum albumin and 0.05 per cent Tween matrix unit corresponds to 1/10 of the maximum 20 as sample diluent and 0.1 M PBs-Tween 20 value obtained with a standard positive serum (0.05 per cent) as conjugate diluent. The substrate sample. The titre of the test sample was taken was 0.04 per cent O-phenylenediamine (OPD) and as the highest dilution showing one matrix unit 0.015 per cent hydrogen peroxide in citrate phos- above the value obtained in the lowest dilution phate buffer (pH 5). After each step plates were of standard negative sample (1:4).

74

R. Sharma, Z. Woldehiwet

Antibody capture assay For the ACA, ELISA plates were coated with optimally diluted c~-chain specific swine antisheep IgA. The plates were-then incubated with serial fourfold dilutions of test serum samples or controls, followed by BRSVantigen, anti-BRSV mAb, biotinylated anti-mouse IgG, ABCcomplex (ABC kit, Sera Laboratories) and substrate solution, in that order, as described earlier in the ~I~ASassay. Biotinylated antibody and ABC complex were used according to manufacturers' instructions.

TABLE 1: Lymphocyte subpopulations in peripheral blood of lambs reinfected with bovine respiratory syncytial virus

Days after infection 6

5

Clinical observations Lambs experimentally reinfected with bovine Rsv showed mild clinical signs characterised by serous nasal discharges between three and five days after infection. Coughing was observed in only two lambs six days after infection and pyrexia was not observed in any of the lambs reinfected with bovine e.sv. No adventitious lung sounds were heard in any of the lambs.

Virological studies Bovine Rsv was isolated from nasal swabs obtained from three of eight lambs five days after reinfection. The virus was also isolated from the mononuclear fraction of peripheral blood obtained from two of the three virus-positive lambs five days after reinfection.

Lymphocyte subpopulations The proportions of lymphocyte subpopulations were drastically affected after challenge. Five days after challenge, the percentages of peripheral blood T (CD5+) lymphocytes were significantly reduced from 36.59 + 0-94 to 21-73

RSV

RSV Control

Analysis of data

Results

Group

Control

10

The Student's t test was. used to compare mean values obtained from lambs experimentally reinfected with bovine Rsv and those obtained from control uninfected lambs. The paired t test was used to compare values obtained before infection with values obtained after experimental infection.

Lymphocyte subpopulations (mean +- SE percentage)

RSV Control

14

RSV Control

CD5+ LCA p220+ CD4+

CD8+

CD4CD8-

36"59 _+0'94 36.46 _+0"83

53"06 +-2"60 55"89 _+1-90

19"70 +-1"70 21 "96 +-1"69

8"57 8'08 +-0"51 _+1"54 8"88 7.30 +-1.04 +-1.50

21 "73 _+2"70 37.75 +0"88

74"16 _+3"07 56.36 +3.10

12'99 13'69 + 2 " 3 0 +1'12 22-52 9"26 +2"19 +0"69

30"61 +1 "30 40.76 •+2.90

62"55 +-1"90 49"37 _+2"80

16'60 10"44 4"40 + 0 " 5 3 +0"82 +1 "37 21"30 8"08 12-07 +-2.25 +-1.02 +-4.28

31 "99 +-1"30 40.19 +2-40

66"19 +-1"46 50"07 _+1.90

15"96 8"41 + 0 " 9 9 +0"81 18,68 8"58 +-2.12 + 1 . 3 5

0'63 +-0"61 7"10 -+2.02

7"61 +1"00 12-25 +-3.36

+ 2.70 (P<0-01) (Table 1). The decrease in CD5+ lymphocytes continued until 14 days after the challenge. The proportions of B lymphocytes were significantly increased from 53.06 _+ 2.60 per cent to 74.16 + 3.07 per cent five days after challenge (P<0.001) and remained at higher levels up to 14 days after challenge (P<0-01) (Table 1). The proportion of CD4+ T lymphocytes was reduced from 19.71 _+ 1.70 per cent before inoculation to 12.99 + 2-30 per cent five days after challenge (P<0-01) (Table 1). The CD4+ lymphocytes remained at low levels up to 10 days after challenge (P<0-05) (Table 1). The proportions of CD8+ lymphocytes were increased significantly five days after inoculation (P<0.01) but returned to pre-inoculation levels 10 days after challenge (Table 1). The proportions of lymphocytes with the pan T cell marker (CD5+) but without markers for CD4 or CD8 (CD4-CD8-) were significantly reduced from 8.08 + 1.54 per cent to 0.63 + 0.61 per cent five days after challenge (P<0.05) (Table 1). There were no significant changes in lymphocyte subsets in the peripheral blood of control lambs.

Lymphocyte transformation responses All the results are the mean of triplicate cultures. There were no significant differences in the uptake of tritiated thymidine between cultures

Reinfection of lambs with bovine respiratory syncytial virus from control and bovine RSv-infected lambs when incubated without PHA or bovine Rsv antigen. The mean stimulation index in PHA stimulated cultures of infected lambs was significantly reduced from 45.54 + 14.8 before challenge to 9.94 _+ 5-67 five days after reinfection (P<0.01). The stimulation index of lymphocytes obtained from control animals five days after inoculation was also reduced but was significantly higher than that of lymphocytes obtained from RSVinfected lambs. In Rsv-infected animals the mean stimulation index remained significantly depressed until 10 days after reinfection (P<0.05) followed by slight recovery 14 days after reinfection (Fig 1). The mean stimulation index of RSV antigenstimulated cultures was significantly increased from 1.47 _+0-32 before challenge to 3.46 + 1.64 five days after challenge (P<0.01). Rsv antigeninduced LT responses continued to be detected until 14 days after infection (Fig 1).

75

5

o'~ O

-El- IgA - A - IgM ~ IgG

4

3

1

-~ c

2

rr 1

0

0

-~

t:~

2i

Days after inoculation FIG 2: Mean titres of RSV-Specific IgM, IgG and IgA in serum samples obtained from eight lambs reinfected with bovine Rsv

from the corresponding titres in serum samples obtained 14 and 21 days after primary infection (P>0-05).

Class-spec~'c antibody responses Before challenge, all lambs previously infected with bovine RSV had low titres of virus-specific IgG but had no detectable virus-specific IgM or IgA. Ten days after challenge, there was a significant increase in virus-specific IgG, IgM and IgA (Fig 2) (P<0.01). Virus-specific IgG titres were significantly higher than IgM and IgA titres throughout (P<0.01). Virus-specific antibody titres in serum samples obtained 14 and 21 days after challenge were not significantly different 70 (3 • [] []

60

~,

50

"-

40

O -~ E

RSVc RSVt PHAc PHAt

30 20

0~

I

t

I

I

0

5

10

14

D a y s after i n o c u l a t i o n FIG 1 : Lymphocyte transformation responses to bovine Rsv antigen and to the mitogen phytohaemagglutinin (PHA) in lambs reinfected with RSV (RSVt, PHAt) and in control lambs (RSVc, PHAc)

Discussion

Reinfection with RSVis a common event, occurring in 40 to 50 per cent of human adults in close contact with patients infected with human RSV, despite the presence of complement fixing and neutralising antibodies following previous infection (Ward et al 1983, Openshaw et al 1988). Reinfection with bovine RSVhas also been reported in calves but it is not accompanied by clinical signs of disease (Martin 1983, Kimman 1989). Children may exhibit severe clinical disease after challenge but the severity of clinical disease is reported to be progressively reduced after subsequent exposures (Henderson et al 1979). In the present study, challenge of lambs was accompanied by very mild clinical signs, with only two of eight lambs showing serous nasal discharge and coughing. Reinfection of lambs with bovine RSVwas confirmed by isolation of virus from three of eight nasal swabs and seroconversion in the other lambs. Two of the three virus-positive lambs had viraemia as demonstrated by isolation of virus from mononuclear cell fractions. Several workers have shown that calves can be reinfected with bovine RSV (Verhoeff 1983, Martin 1983, Stott and Taylor 1985, Kimman et al 1987a) but the rate of virus shedding in calves after challenge

76

R. Sharma, Z. Woldehiwet

is not well established. For example, Kimman et al (1987a) did not isolate virus from calves experimentally reinfected with bovine RSV but Stott et al (1984) recovered virus after challenge from 14 of 16 calves vaccinated with two live virus vaccines. On the other hand, virus has been regularly recovered from human infants naturally infected with RSV (Beem 1967, Henderson et al 1979, Fernald et al 1983). Because of the difficulty in reproducing experimental bovine Rsv infection in calves, studies on the effect of challenge on cellular immune responses are lacking. In the present study, it was demonstrated that reinfection of lambs was accompanied by a significant rise in B lymphocytes and CD8+ lymphocytes but there were significant reductions in the proportions of CD4+ and CD4-CD8 (CD5+) T lymphocytes. The reductions in CD4+ and CD4-CD8- T cells and the increases in CD8+ T cells following reinfection were similar to those observed in lambs after primary infection with bovine RSV (Sharma et al 1990) but the increase in B cells is in sharp contrast to the reductions observed after primary infection (Sharma et al 1990). Similar reductions in T helper cells have also been reported in recurrent respiratory tract infection (Baranda et al 1984) probably attributable to human RSV. The significant stimulation of lymphocytes by bovine Rsv antigen in the mononuclear cells obtained from lambs after challenge is in sharp contrast to the lack of any response in cells obtained after primary infection (Sharma and Woldehiwet 1990c). In human infants some workers have shown increased lymphocyte reactivity to RSV antigen following repeated infections (Fernald et a11983). Schaufet al (1979) suggested that a single Rsv infection does not always confer enough cellular immunity to be demonstrable in lymphocyte transformation assays. Sharma and Woldehiwet (1990c) showed that lymphocyte responses to the mitogen PHAwere severely affected five days after primary infection. These reductions were attributable to the reductions in numbers and reactivities of CD4+ cells (Sharma and Woldehiwet 1990c). Lymphocytes obtained after challenge were also significantly less responsive to PHA.The depressed lymphocyte transformation responses coincided with reductions in CD4+ cells. The patterns of antibody responses after challenge are suggestive of an anamnestic response. Kimman et al (1987a) demonstrated rapid and

high virus-specific IgG and IgA responses in four colostrum-deprived calves after challenge with bovine RSVbut they could not demonstrate virusspecific IgM in any of the calves. On the other hand, in human infants, Welliver et al (1980) found accelerated responses in all three antibody classes. Virus-specific IgG was the predominant antibody after challenge. A similar boost in virusspecific IgG has been recorded in immune subjects exposed to measles, mumps, rubella and influenza A viruses (Stokes et al 1961, Brunell et al 1968, Davies et al 1971, Frank et al 1979). The rapid and increased antibody responses in lambs coincided with increased proportions of B cells after challenge.

Acknowledgements The authors wish to thank Dr F. Westenbrink, Central Veterinary Institute, Lelystad, The Netherlands, for providing anti-BRSVmonoclonal antibodies and Dr M. R. Brandon, University of Melbourne, Australia, for providing the monoclonal antibodies to identify lymphocyte subsets. R. S. is a Commonwealth scholar supported by the Association of Commonwealth Universities.

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Veterinary Science fi0, 152-156 SHARMA, R., WOLDEHIWET, Z., SPILLER, D. G. & WARENIUS, H. M. (1990) Lymphocyte subpopulalions in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus. Veterinary Immunology and lmmunopathology 24, 383-391 STOKES, J. Jr, REILLY, C. M., BUYNAK, E. B. & H1LLEMAN, M. R. (1961) Immunologic studies of measles. American Journal of Hygiene 74, 293-303 STOTT, E. J. & TAYLOR, G. (1985) Respiratory syncytial virus: brief review. Archives of Virology 84, 1-52 STOTT, E. J., THOMAS, L. H., TAYLOR, G., COLLINS, A. P., JEBBETT, J. & CROUCH, S. (1984) A comparison of three vaccines against respiratory syncytial virus in calves. Journal qf Hygiene (Cambridge) 93, 251-261 VERHOEFF, J. (1983) Some aspects of respiratory viral infections in dairy cattle. Doctoral thesis, University of Utrecht WARD, K. A., LAMBDEN, P. R., OGILVIE, M. M. & WATT, P. J. (1983) Antibodies to respiratory syncytial virus polypeptides and their significance in human infection. Journal of General Virology 64, 1867-1876. WELLIVER, R. C., KAUL, T. N., PUTNAM, T. I., SUN, M.. RIDDLESBERGER, K. & OGRA, P. L. (1980) The antibody response to primary and secondary infection with respiratory syncytial virus infection: kinetics of class-specific responses. Journal of Pediatrics 96, 808-813 Received October 29, 1990 Accepted September 2, 1991