Immune responses of lambs experimentally infected with bovine respiratory syncytial virus and Pasteurella haemolytica

j. Comp. Path. 1991 Vol. 105

Immune Responses of Lambs Experimentally Infected with Bovine Respiratory Syncytial Virus and Pasteurella haernolytica R. S h a r m a a n d Z. W o l d e h i w e t * University of Liverpool, Department of Veterinary Pathology, VeterinaryField Station, Leahurst, Neston, Wirral, L64 7TE, U.K.

Summary The immune responses of lambs experimentally infected with bovine respiratory syncytial virus (RSV) and Pasteurella haem@tica were compared with lambs infected with bovine RSV or Pasteurella haemolytica alone. Superinfection with P. haemolytica aggravated the reduction in the number of CD5 +, CD4 + and LCA p220 + (B) lymphocytes caused by bovine RSV, but it had no effect on the neutralizing antibodies against P. haemolytica cytotoxin. Serum samples obtained from lambs experimentally infected with bovine RSV and P. haemolylica had significantly lower amounts of neutralizing antibodies to bovine RSV than those obtained from lambs infected with bovine RSV alone.

Introduction Lambs experimentally infected with bovine respiratory syncytial virus (bovine RSV) are significantly more susceptible to secondary infection with Pasteurella haemolytica (A1-Darraji, Cutlip, Lehmkuhl and Graham, 1982a; A1-Darraji, Cutlip, Lehmkuhl, Graham, Kluge and Frank, 1982b; Trigo, Breeze, Liggitt, Evermann and Trigo, 1984; Sharma and Woldehiwet, 1990a). The increased susceptibility to secondary bacterial infections following some viral infections is thought to be caused by non-specific immunosuppression (Filion, McGuire and Babiuk, 1983). Infection with bovine RSV is characterized by significant alterations in lymphocyte subpopulations in lambs (Sharma, Woldehiwet, Spiller and Warenius, 1990) and mononuclear cells obtained from lambs experimentally infected with bovine RSV are more susceptible to P. haemolytica cytotoxin and less responsive to mitogenic blastogenesis (Sharma and Woldehiwet, 1990a, 1991a). In the present study we examined the effects of dual infections with bovine RSV and P. haemolytica on the immune responses of lambs to bovine RSV and to the cytotoxin of P. haemolylica.

* Corresponding author. 0021-9975/91 •060157 + 10 $03.00/0

© 1991 AcademicPress Limited

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R. Sharma and Z. Woldehiwet Materials and Methods

Bovine Respiratory Syncytial Virus A strain of bovine RSV (BRSV 66), passaged in lamb testis cells (LTC) three times, was maintained and used as previously described (Sharma and Woldehiwet, 1990b).

Bacterial Inoculum A reference strain of P. haemolytica biotype A serotype 1 was obtained from the Moredun Institute, Edinburgh. The 1-hour log phase cultures of P. haemolytica were prepared as described by Woldehiwet and Rowan (1988).

Lambs Thirty-two 6- to 8-week-old conventionally reared Suffolk crossbred iambs were divided into four groups. One group of eight lambs was inoculated with bovine RSV alone. Another group of eight lambs was infected with bovine RSV followed with P. haemolytica 6 days later as described earlier (Sharma and Woldehiwet, 1990a). A third group of eight Iambs was infected with P. haemolytica alone and a fourth group &eight control lambs was inoculated with sterile cell culture fluid and, 6 days later~ with sterile bacterial culture medium.

Collection of Blood Samples Peripheral blood samples were collected from all the groups of lambs in EDTA-coated tubes on days - 6 , - 1 , 1, 5, 11, 16 and 21 post-inoculation (P.I.) with P. haemolytica. These samples were used to establish the total leucocyte counts with a Coulter Counter (Coulter Electronics Ltd, U.K.) and differential leucocyte counts after staining blood smears with Giemsa. Heparinized samples, collected on - 6 , - 1, 1, 1, 5, 11, 16 and 21 days P.I. with P. haemolytica, were used to establish the distribution of lymphocyte subpopulations and lymphocyte transformation (LT) responses. Mononuelear cells (MNC) were separated from diluted blood on a Ficoll-Paque column (Pharmacia Fine Chemicals, Uppsala, Sweden). Cell viability was determined by the trypan blue exclusion technique. Changes in the number of lymphocyte subsets following infection were established by flow cytometry with monoclonal antibodies kindly provided by Dr M. R. Brandon, University &Melbourne, Australia, as described previously (Woldehiwet and Sharma, 1990).

Lymphocyte Transformation Assays Lymphocyte transformation assays were performed on MNC obtained from eight lambs infected with P. haemolytica, eight lambs infected with bovine RSV followed by P. haemo~tica and eight control lambs on - 5 , - 1 , 5, 11 and 16 days P.I. with P. haemolytica with 20 txl of purified phytohaemagglutinin (40 Ixg per ml) (Wellcome Reagents Ltd., Beckenham, Kent, U.K.) or 20 I~1 of optimum concentration of heat inactivated P. haemo~tica antigen, bovine RSV antigen or 20 ~tl of RPMI 1640 medium as described earlier (Sharma and Woldehiwet, 1991a). Cultures were incubated at 370C in a humidified atmosphere containing 5 per cent CO 2 in air for 72h. Radiolabelling was carried out by adding 1 IxCi of [3H]-thymidine (Radiochemical Centre, Amersham, U.K.) per well in 201.d of RPMI 1640 medium 24h before terminating incubation. The degree of transformation was expressed as the ratio of [3H]-thymidine incorporation in cultures stimulated by phytohaemagglutinin (PHA), P. haemolytica antigen or bovine RSV antigen to that of the control cultures (stimulation index, SI).

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Virus Neutralizing (VN) Antibodies Serum samples collected 0, 11, 16 and 21 days after inoculation with bovine RSV were examined for neutralizing antibodies to bovine RSV as described earlier (Sharma and Woldehiwet, 1990b).

Cytotoxin Neutralizing Antibodies Serum samples, obtained sequentially from lambs inoculated with bovine RSV and P.

haemolytica or P. haemolytica alone on days - 1 , 5, 11, 16 and 22 after P. haemolytica inoculation, were analysed for eytotoxin neutralizing antibodies. P. haemolytica Cytotoxin

P. haemolytica cytotoxin was prepared, and titrated by the MTT dye (3-[4,5dimethylthiazoyl-2-yll-2,5-diphenyltetrazolium bromide, Sigma Chemicals Co. Ltd., Poole, Dorset, U.K.) reduction test (Sbarma and Woldehiwet, 1990a). The units of activity contained in 0"2 ml were defined as the reciprocal of the dilution causing 50 per cent cytotoxicity and the units of activity contained in 1 ml of undiluted cytotoxin were calculated by multiplying this value by 5.

Cytotoxin Neutralization Test The cytotoxin neutralization titres were determined by the M T T dye reduction assay as described by Vega, Maheswaran, Leininger and Ames (1987). Briefly~ 100 gl of serial two-fold dilutions of test serum sample were prepared in triplicate in 96-well flat-bottom microtitre plates. One hundred gl ofcytotoxin, containing 30 units per ml, was added to each well and the plates were incubated for 1 h at 37°C. After incubation, 100 gl of peripheral blood mononuclear cells (PBMNC) (5 x 106 cells per ml), prepared from normal healthy lambs were added to each well. The following controls were included in each test: (1) for eytotoxicity (cytotoxin+ RPMI medium+ PBMNC); (2) for serum-related cytotoxicity (serum+RPMI medium+PBMNC); (3) for cell viability ( P B M N C + R P M I medium); (4) for non-specific dye reduction (RPMI medium only). After incubation for 1 h at 37°C, 20 I~1 of MTT dye at a concentration of 5 mg per ml was added to each well and the plates were incubated for a further 4h. One hundred ~1 of acid isopropanol (0.04 N HC1 in isopropanol) containing 3 per cent sodium dodecyl sulphate was then added to each well and the absorbance of each well determined in a MR 700 Microplate Reader (Dynatek Laboratories Inc. Alexandria, Virginia, U.S.A.) at a test wavelength of 570 nm, a reference wavelength of 630 nm and a calibration setting of 1"00. The tests were validated with appropriate controls for non-specific dye reduction. Percentage of neutralization was calculated as follows: ODxODy xl00 Neutralization (per cent) = 1 OD x'-OD y' where OD x = O D of serum-treated cells; OD y = O D of cytotoxin plus test serumtreated cells; OD x ' = OD of untreated control cells; OD y'= OD of cytotoxin treated cells. The reciprocal of the highest serum dilution that caused 50 per cent neutralization was considered as the neutralization titre.

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Analysis of Data The Student's t-test was used to compare mean values obtained from lambs experimentally infected with bovine RSV and P. haemolytieawith those obtained from control uninfected lambs or corresponding data obtained from Iambs inoculated with either of these agents alone. The paired t-test was used to compare values obtained before infection with values obtained after experimental infection. Results

Lambs experimentally infected with bovine RSV and P. haemolytica showed severe respiratory signs characterized by dullness, nasal discharge, coughing, significant pyrexia, high disease scores and high mortality rates as previously described (Sharma and Woldehiwet, 1990a). The alterations in lymphocyte subpopulations of lambs experimentally infected either with bovine RSV or with P. haemo~tiea alone have been described elsewhere (Sharma et al., 1990; Sharma and Woldehiwet, 1991b). Infection of lambs with bovine RSV followed by a superinfection with P. haemolytica 6 days later was characterized by even more severe alterations in lymphocyte subpopulations. Five days after infection with bovine RSV, the numbers of CD5 +, CD4 + and LCA p220 + (B) lymphocytes were significantly reduced (P<0.01, P<0"002 and P<0"05, respectively) (Figs 1 and 2). Superinfection with P. haemolytica resulted in further reductions in the number of CD5 + lymphocytes for a further 24 h, and a reduction in the numbers of CD4 + T lymphocytes and B lymphocytes for a further 5 days (Figs 1 and 2).

Lymphocyte Transformation Responses All the results are the means of triplicate cultures. There were no significant differences in the uptake of tritiated thymidine between cultures from control and bovine RSV and P. haemolytica-infected lambs when incubated without PHA or bovine RSV antigen or P. haemolytica antigen. There were animal to animal and day to day variations in the transformation responses of lymphocytes to PHA in both control and infected lambs. The variations observed in the control lambs were not statistically significant (Fig. 3) ( P > 0"05). However, the mean stimulation index in PHA stimulated cultures of infected lambs was significantly reduced from 38.174-7"11 before inoculation to 9"254-2"7 five days after bovine RSV infection (Fig. 3) (P<0'01). The mean stimulation index was further reduced to 7"06 4- 1"29 on day 5 after bacterial superinfection ( P < 0.01) and remained depressed until 16 days after bacterial infection. Bovine RSV antigen-induced and P. haernolytica antigen-induced transformations were not observed in any of the MNC cultures obtained from bovine RSV and P. haemolytica-infected lambs and control lambs on different days after inoculation.

Virus Neutralizing Antibodies Low levels of virus neutralizing (VN) antibodies were detected in all lambs infected with bovine RSV 11 days P.I. High titres occurred 16 to 21 days after

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Cytoloxin Neutralizing Antibodies Prior to inoculation, low titres of cytotoxin neutralizing antibodies were detected in three lambs that later were infected with both the agents, but 16 days after inoculation with P. haemolytica there was a significant rise in cytotoxin neutralizing antibodies in all eight lambs (Fig. 5) (P<0"01). A further increase in the titres occurred 22 days P.I. Cytotoxin neutralizing antibodies were detected at significant levels in lambs infected with P. haemolytica alone 14 days P.I. There were no significant differences in the titres of cytotoxin neutralizing antibodies between the two groups of lambs (P> 0"05). i

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Discussion

It has been shown that the enhanced susceptibility of lambs infected with bovine RSV to subsequent challenge with P. haemolytica is characterized by increased mortality rates and enhanced bacterial growth (Sharma and Woldehiwet, 1990a). Several other workers have also produced clinical and pathological data to demonstrate increased susceptibility of bovine RSV-infected lambs to P. haemolytica infection (A1-Darraji et al., 1982a,b; Trigo et al., 1984) but the reasons for this enhanced susceptibility are not clear. Previous studies on viral-bacterial synergism in respiratory infections ['avour the notion that virus infections reduce the efficiency of the lungs in clearing bacteria and depress the phagocytic or bactericidal potential of alveolar macrophages (Woolcock, 1979) but there is some evidence which suggests that alveolar macrophages are not permissive to the replication of bovine RSV and that macrophages exposed to bovine RSV remain functionally normal (Trigo, Liggitt, Evermann, Breeze, Huston and Silflow, 1985; Roberts, 1982).

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We had previously demonstrated alterations in lymphocyte subpopulations in the peripheral blood of lambs 10 days after experimental infection with bovine RSV (Sharma et al., 1990). In the present study, we demonstrated that the alterations in lymphocyte subpopulations that accompany infection with bovine RSV occur 5 to 10 days after infection. This period coincided with the period of increased susceptibility to superinfection with P. haern@tica (Sharma and Woldehiwet, 1990a). Previous studies have shown that two factors are critical in the induction of pasteurellosis; viral exposure and the timing of the bacterial superinfection (Filion et al., 1983). A1-Darraji el al. (1982b) demonstrated that the clinical signs were more pronounced in those lambs inoculated with P. haemolytica 5 days after infection with bovine RSV than in those inoculated with P. haemolytica 3 days after infection with bovine RSV. The alterations in lymphocyte subpopulations owing to bovine RSV persisted for longer periods when lambs were superinfected with P. haemolytica. The reductions-in the numbers of CD5 + and CD4 + lymphocytes, attributable to P. haemolytica alone, are transient (Sharma and Woldehiwet, 1991b) compared with the sustained reduction in lambs infected with both agents. Infection with P. haemolytica alone also resulted in transient depression of lymphocyte transformation responses to PHA compared to the prolonged depression in lambs infected with bovine RSV and P. haemolytica. These findings could explain the severity of the clinical signs and the extent of pneumonia in lambs infected with both the agents compared to those inoculated with either of the agents alone (A1 Darraji et al., 1982a, b; Sharma and Woldehiwet, 1990a). Kennedy-Stoskopf, Narayan and Hirsch (1983) also demonstrated significant reductions in LT responses to PHA and concanavalin A (Con A) in lymphocytes obtained from goats 3 to 5 days after infection with PI-3 virus. They suggested that this might be an important mechanism of viralbacterial synergism between PI-3 virus and P. haemolytiea. An attempt was made to study whether infection with bovine RSV and P. haemolytica affected the development of humoral antibodies to bovine RSV or to P. haemolytica cytotoxin. Significant increases in cytotoxin neutralizing antibodies were detected 14 to 16 days after infection with P. haemolyticain both groups of lambs. There were no significant differences in the titres ofcytotoxinneutralizing antibodies in serum samples obtained from lambs infected with P. haemolytica and those obtained from lambs infected with bovine RSV and P. haemolytica. It appears that bovine RSV does not affect the development of humoral immunity to the cytotoxin once the animals have survived the early days of superinfection. Filion et al. (1983) reported that, in calves, the immunosuppression induced by Bovine herpes virus-1 (BHV-1) did not appear to interfere with the humoral immune responses to the virus or to P. haemolytica. On the contrary, VN antibody titres against bovine RSV in lambs infected with bovine RSV and P. haemolytica were significantly lower than those in lambs infected with bovine RSV alone. This suggests that P. haernolytica superinfection may interfere with the development of neutralizing antibodies to bovine RSV. Acknowledgments The technical assistance of C. Savage and A. Jopson is gratefully acknowledged. We

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would like to thank Dr M. R. Brandon, University of Melbourne, Australia for providing the monoclonal antibodies. R. Sharma was a Commonwealth Scholar. References

A1-Darraji, A. M., Cutlip, R. C., Lehmkuhl, H. D. and Graham, D. L. (1982a). Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: pathologic studies. American Journal of VeterinaryResearch, 42, 224-229. A1-Darraji, A. M., Cutlip, R. C., Lehmkuhl, H. D., Graham, D. L., Kluge, J. P. and Frank, G. H. (1982b). Experimental infection of lambs with bovine respiratory syncytial virus and Pasteurella haemolytica: clinical and microbiologic studies. American Journal of Veterinary Research, 42, 236-240. Filion, L. G., McGuire, R. L. and Babiuk, L. A. (1983). Non-specific suppressive effect of bovine herpesvirus type 1 on bovine leucocyte functions. Infection and Immunity, 42, 106-112. Kennedy-Stoskopf, S., Narayan, O. and Hirseh, R. L. (1983). Immunosuppression in goats inoculated with PI-3 virus. American Journal of Veterinary Research, 44, 2302-2306. Roberts, N. J. Jr. (1982). Different effects &influenza virus, respiratory syncytial virus, and sendai virus on human lymphocytes and macrophages. Infection and Immunity, 35, 1142-1146. Sharma, R. and Woldehiwet, Z. (1990a). Increased susceptibility to Pasteurella haemolytica in lambs experimentally infected with bovine respiratory syncytial virus. Journal of Comparative Pathology, 103, 267-272. Sharma, R. and Woldehiwet, Z. (1990b). Pathogenesis of bovine respiratory syncytial virus in experimentally infected lambs. Veterinary Microbiology, 23, 267-272. Sharma, R. and Woldehiwet, Z. (1991a). Depression of lymphocyte responses to phytohaemagglutinin in lambs experimentally infected with bovine respiratory syncytial virus. Research in Veterinary Science, 50, 152-156. Sharma, R. and Woldehiwet, Z. (1991b). Effects of Pasteurella haemolytica on the lymphocyte subpopulations in the peripheral blood of lambs. Veterinary Microbiology, 27, 159-168. Sharma, R., Woldehiwet, Z., Spiller, D. G. and Warenius, H. M. (1990). Lymphocyte subpopulations in peripheral blood of lambs experimentally infected with bovine respiratory syncytial virus. VeterinaryImmunology and Immunopathology, 24, 383-391. Trigo, F. J., Breeze, R. G., Liggitt, H. D., Evermann, J. F. and Trigo, E. (1984). Interaction of bovine respiratory syncytial virus and Pasteurella haemolytica in the ovine lung. American Journal of Veterinary Research, 45, 1671-1678. Trigo, E., Liggitt, H. D., Evermann, J. F., Breeze, R. G., Huston, L. Y. and Silflow, R. (1985). Effect of in vitro inoculation of bovine respiratory syncytial virus on bovine pulmonary alveolar macrophage function. American Journal of Veterinary Research, 46, 1098-1103. Vega, M. V., Maheswaran, S. K., Leininger, J. R. and Ames, T. R. (1987). Adaptation of a colorimetric microtitration assay for quantifying Pasteurella haemolytica A1 leukotoxin and antileukotoxin. American Journal of Veterinary Research, 48, 1559-1564. Woldehiwet, Z. and Rowan, T. G. (1988). Effects of environmental temperature, relative humidity and vaccination on Pasteurella haemdytica in lungs of mice. Journal of Comparative Pathology, 98, 433-439. Woldehiwet, Z. and Sharma, R. (1990). Alterations in lymphocyte subpopulations in peripheral blood of sheep persistently infected with border disease virus. Veterinary Microbiology, 22, 153-160. Woolcock, J. B. (1979). Bacterial infection and immunity in domestic animals. Developments in Animal and Veterinary Sciences, 3, 87-98.

I Received, October 27th, 1990-] Accepted, March 29th, 1991 _]