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).

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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

Reinfection of lambs with bovine respiratory syncytial virus.

Eight lambs which were experimentally infected with bovine respiratory syncytial virus (RSV) when they were six to eight weeks old were challenged wit...
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