Effect of BVD virus infection on alveolar macrophage functions

Veterinaru

and

Veterinary Immunology and Immunopathology 46(1995)195-210

immunopathology

Effect of BVD virus infection on alveolar macrophage functions M.D. Welsh*, B.M. Adair, J.C. Foster Veterinary Sciences Division, Stormont, Belfast BT4 3SD, UK

Accepted 27 July 1994

Abstract Alveolar macrophages (AM ) were recovered by bronchoalveolar lavage from a group of eight calves at various times before and after inoculation with a cytopathic respiratory isolate of bovine viral diarrhoea virus (BVDV). A second group of four calves were given tissue culture medium as a control inoculum. Macrophages were also recovered from two additional, uninoculated calves, and were exposed to BVDV in vitro. Tests were carried out on the recovered macrophages to determine the effects of the virus on several functional properties. Immunofluorescence did not indicate the AM as being readily susceptible to this isolate of BVDV, although infection did occur. Fc receptor (FcR) and complement receptor (C3R) expression, phagocytosis and microbicidal activity and the production of neutrophi1 chemotactic factors were all significantly reduced in macrophages recovered from BVDV infected calves, compared with pre-inoculation control levels, whereas the control inoculated calves displayed significant increases in some of the functions. With macrophages exposed to the virus in vitro however, only FcR and C3R expression and phagocytic activity were significantly reduced. The results demonstrate that BVDV can reduce local immune defences in the lung, following infection by the respiratory route, and in conjunction with the other immunosuppressive properties of BVDV would favour a pre-disposing role for the virus in the pathogenesis of respiratory disease in calves. Abbreviations AM = Alveolar macrophages; BHV- 1 = Bovine herpesvirus type- 1; BVDV = Bovine viral diarrhoea C3R = Complement receptors; FcR = Fc receptors: virus: l

Corresponding author: Tel. + 44 232 5200 I 1; Fax + 44 232 76 1662.

0165-2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO1652427(94)05366-9

196

M.D. Welsh et al. / Veterinary Immunology and Immunopathology 46 (1995) 195-210

IF= Immunofluorescence; E-2; pi = Post-infection; virus.

NCF= Neutrophil pi-3 =Parainfluenza

chemotactic factors; PGE-2 = Prostaglandin type-3 virus; RSV= Respiratory syncytial

1. Introduction

A relationship between BVDV and respiratory disease has been recognised for some time (Barker, 1990), and BVDV along with parainfluenza-3 virus (PI-3) and respiratory syncytial virus (RSV) have been the viruses most frequently recovered from outbreaks of calf pneumonia (Bryson, I985 ) . Also BVDV has been the viral agent most often isolated from pneumonic lungs in cases of shipping fever (Reggiardo, 1979 ). Although experimental reproduction of respiratory disease with BVDV has produced conflicting reports (Radostits and Townsend, 1989 ), its involvement in field cases may go severely under diagnosed since the presence of lesions does not always correlate with clinical disease (Reggiardo, 1979). The alveolar macrophages (AM) play a vital role in protecting the lungs against infection, and provide an important first line of defence against respiratory pathogens (Khadom et al., 1985; Fels and Cohn, 1986 ). Impairment of AM functions after respiratory viral infection has been associated with predisposition to secondary pulmonary infection (Morahan et al., 1985 ), and up to 90% of bacterial pneumonias are thought to develop following viral infections (Babiuk et al., 1988). Although it is conceivable that BVDV would be involved in respiratory disease due to its immunosuppressive properties (Potgieter, 1988), there may also be a case for a more direct involvement of BVDV in respiratory disease by direct infection of the respiratory system. Therefore, an understanding of the effects of BVDV on AM functions (local respiratory immunity) is essential if the role of the virus in respiratory disease is to be fully understood. To date this has not been studied in any detail. In this paper the effects of BVDV on certain functional properties of AM following in vivo and in vitro infection are investigated.

2. Materials and methods 2.1. Virus and cell culture

Semi-continuous foetal calf lung (FCL) cells (BVDV free) were grown in baby hamster kidney 21 (BHK) medium containing 10% heat-inactivated foetal calf serum (HIFCS). BHK containing 2% HIFCS was used for maintenance of the cell cultures. A cytopathic BVDV isolate ( 1874) recovered from the lungs of a calf which had died from pneumonia was propagated in FCL cells. The virus had been pas-

M.D. Welsh et al. / Veterinar_vImmunology and 1nmunopatholog.v 46 (1995) 195-210

197

saged six times in FCL cells prior to use. A virus pool was prepared by inoculating FCL monolayers grown in 75 cm2 plastic flasks at a multiplicity of infection (m.o.i. ) of 0.2 TCID,O per cell. When monolayers showed 70-90% CPE the cells were frozen and thawed three times, and the cell debris was removed by centrifugation (500 xg, 10 min). The infectivity titre of this pool was 1O6TCIDso ml- ’ , as measured by endpoint titration in FCL cells in microtitre plates. A control inoculum was prepared from FCL cell monolayers, mock infected with BHK medium containing 2% HIFCS, then incubated and processed in exactly the same manner as the virus pool. 2.2. Immunojluorescence BVDV antigen was detected by direct immunofluorescent (IF) staining of acetone fixed cells. Bovine and rabbit polyclonal antisera for direct IF detection of BVDV, RSV and PI-3 virus were conjugated with fluorescein isothiocyanate (FITC) and used as described previously (McNulty and Allan, 1984). Slides were mounted using Citifluor (Citifluor Ltd., London) and examined by UV microscopy. 2.3. Experimental animals and experimental design Twelve conventionally reared, cross-bred calves were used. Four calves were used as controls, to determine the effects of virus free cell culture medium on macrophage functions. The remaining eight calves were divided into two groups of four, and each group was inoculated with BVDV and subjected to bronchoalveolar lavage on alternate days throughout the experiment. Functional tests were carried out on the AM recovered from each calf. Baseline levels for all tests were determined during an initial 2 week period, prior to inoculation of the calves with control inoculum (FCL lysate) or BVDV. The control group of calves was inoculated with FCL lysate and the two virus groups of calves with BVDV. Each calf was given 10 ml of inoculum intranasally and 10 ml intratracheally on each of two consecutive days, so that each animal received a total of 40 ml of inoculum (virus titre 1O6TCID,, ml- ’ ). For the in vitro experiments, two additional 3-month-old calves were slaughtered, AM were recovered from the lung washings, and exposed to BVDV in vitro as detailed below. All calves were free of current BVDV infection as indicated by direct immunofluorescent staining of FCL cells, 7 days after coverslip cultures were exposed to serum from each animal. Nasal mucus samples were also taken using sterile cotton swabs (two per calf ). The swabs were placed in 5 ml of Eagle’s minimal essential medium (EMEM ) , vortexed for 30 s, and cytospin slide preparations (Shandon Scientific Ltd, Runcorn, UK) made. These were fixed in acetone and tested by direct immunofluorescence for BVDV, RSV and PI-3 virus antigens, as described previously ( McNulty and Allan, 1984). All samples were negative, indicating freedom from current respiratory virus infection. All calves had declin-

198

M.D. Welsh et al. / VeterinaryImmunology and Immunopathology 46 (I 995) 195-210

ing BVDV maternal antibody titres, as indicated by serum neutralisation tests on consecutive serum samples taken at an interval of 2 weeks. 2.4. Alveolar macrophages AM were recovered by bronchoalveolar lavage as described previously (Adair and McNulty, 1992 ). The cells were resuspended in RPM1 1640 medium (G&co, Paisley, UK) containing 50 ,ug ml- ’ gentamicin sulphate (Sigma Chemical Co., Poole, UK) and 10% HIFCS (growth medium), Cell suspensions were prepared containing 5 x 1O5 viable cells ml-‘. Viability was determined by Nigrosin (Sigma) dye exclusion. The AM content of lavage preparations was determined by staining of glass-adherent cells with Diff Quick (Baxter Healthcare, Thetford, UK). AM were also recovered from the lungs of two slaughtered calves. The lungs were excised, filled with normal saline (approximately 1.5 1) and gently massaged to detach loosely adherent cells. The recovered fluid was filtered through sterile gauze, and the cells were centrifuged (3OOxg, 10 min) and resuspended in RPM1 1640 medium containing 50 fig ml-’ gentamicin sulphate and 2.5 pg ml- ’ amphotericin B (Sigma) (RPMI-A). The cell suspensions were then layered onto Ficoll-Paque (Pharmacia, Milton Keynes, UK), and centrifuged at 9OOxg for 40 min at 16°C. The mononuclear cell layer removed from the interface was washed twice in RPMI-A and then the cells were resuspended in medium also containing 10% HIFCS (RPMI-A growth medium ) _AM were infected with BVDV in suspension by centrifuging the cells (300 xg, 10 min) and resuspending in a BVDV suspension at a m.o.i. of 1.O TCIDso per cell, in 500 ml siliconised glass bottles. Control AM suspensions were suspended in FCL cell lysate. Infected and control cultures were incubated for 1 h at 37 ‘C on a shaker, after which the AM were recovered by centrifugation (3OOxg, 10 min) and resuspended in RPMI-A growth medium. 2.5. Culture of alveolar macrophages AM were cultured in four-well plastic multidishes (Nunclon, Roskilde, Denmark). One millilitre of growth medium containing 5 x lo5 macrophages was added to each well. In some experiments sterile 13 mm glass coverslips were added to the wells. The dishes were then incubated for 1 h at 37°C in 5% CO1 to allow the macrophages to settle and adhere. After incubation the cells were washed twice with warm RPMI to remove non-adherent cells. Infected and mock-infected cultures were cultured for 8 days in RPMI-A growth medium. 2.6. Fc and complement receptor assays Fc and C3R expression on virus infected AM were determined by rosetting procedures using antibody and complement opsonised sheep erythrocytes, as described previously (Adair and McNulty, 1992 ) . Counting of Fc and complement

M.D. Welsh et al. / Veterinary ltntnunology and Itntnunopathology 46 (1995) 195-I IO

199

rosettes was carried out following fixation of the AM in methanol for 1 min at room temperature, and staining with Diff Quick (Baxter Healthcare). Coverslips were then mounted in Permount (Fisher Scientific, NJ) and examined using a x40 oil immersion objective. 2.7. Phagocytosis and microbicidal activity Complement-coated Candida krusei cells were used to assess phagocytic and microbicidal activities, as described previously (Adair and McNulty, 1992)) with some minor modifications. In vitro AM cultures used for the C. krusei assays were first washed three times with warm RPM1 to remove the amphotericin B. The viability of the ingested C. krusei cells in the killing assay was determined by staining of the AM with acridine orange (Sigma). Following incubation of the macrophage cultures with the yeast cells, the medium was removed from each well and replaced with 1 ml of normal saline containing 0.1 mg of acridine orange (fresh preparation made daily from x 10 stock solution stored at 4°C). The cultures were incubated for 3 min at room temperature, and the wells were then washed three times with 1 ml volumes of 0.0 1 M phosphate buffered saline (PBS ), pH 7.2. The coverslips were then removed from the wells and rinsed again in PBS, mounted in Citifluor and examined under UV illumination. Viable C. krusei cells were stained green and killed cells stained red/orange. 2.8. Neutrophif chemotactic factors AM were stimulated with opsonised zymosan and the supemates from these cultures were tested for neutrophil chemotactic factors (NCF) using a blind-well chemotactic chamber (Nucleopore Corp., Oxshott, UK), as described previously (Adair and McNulty, 1992 ) . 2.9. Neutralisation tests All serum samples were heat inactivated at 56°C for 30 min before testing. Sera were then diluted in quadruplicate, in two-fold dilutions starting from l/2, using EMEM without serum (50 ~1 volumes). Volumes of virus suspension (50 ~1) in EMEM without serum, containing 100 TCID,, were added to each well containing serum. Virus controls (100, 10, 1.O, and 0.1 TCIDS,,), with 50 ~1 of EMEM replacing serum were included and the plates were incubated at 37 “C in 5% CO1 for 2 h. FCL cells growing in 75 cm2 plastic flasks were trypsinised, washed and re-suspended in EMEM containing 10% FCS at a concentration of 1 x lo5 cells ml- ’ . Volumes of this suspension of 100 ~1 were added to each well. The plates were gently agitated and incubated at 37°C in 5% COZ for 7 days. End point neutralising antibody titres were calculated when the virus controls indicated the presence of 30-300 TCID,, in the test.

200

M.D. Welsh et al. / Veterinary Immunology and Immunopathology 46 (I 995) 195-210

2.10. Statistics

All results were recorded as percentages which were then converted using arcsin transformation before applying a Student’s t-test to determine statistically significant differences between control and virus infected values (Bishop, 198 1). Large variations can occur between separate groups of animals; therefore, to determine significant differences (P-C 0.05 ) statistical comparisons were made between pre- and post-inoculation values for each individual animal. For cultures infected with virus in vitro, statistical comparisons were made between the control and BVDV infected cultures. Error bars representing standard deviations were calculated from the average values obtained for each animal in a particular group. 3. Results 3.1. BVD V infection Clinical signs were not observed in any of the control or BVDV inoculated calves, and no obvious signs of pneumonia were present on gross PM examination of four calves, carried out at the termination of the experiment. All virus inoculated calves showed a substantial (over four-fold) increase in circulating BVDV antibodies as indicated by neutralisation tests (Table 1) carried out 1 day before infection and 4 weeks PI. 3.2. Cell populations The lavage cell populations of the BVDV groups of calves contained more than 80% AM (as indicated by their morphological appearance following staining), and this value remained fairly constant throughout the experiment (pre and post infection). However, the control inoculated calves displayed a slightly lower perTable I Pre and post BVDV infection neutralising antibody titres Calf

1 2 3 4 5 6 I 8

4 weeks post-infection

Pre-infection 2 weeks

1 day

64” 128 64 16 38 76 256 304

54 76 54 76 16 38 153 128

“Reciprocal of 50% end point neutralising antibody titres.

> 1024 > 1024 512 1024 512 > 1024 > 1024 > 1024

M.D. Welsh et al. / Veterinary Itnmunology and Immunopathology 46 (1995) 195-210

90

I

201

_

DAYS POST-INFECTION

100 90

”

1

2

3

4

6

8

DAYS POST-INFECTION

Fig. 1. The percentageof AM expressing FcR following in vivo (A) and in vitro (B) control or BVDV infection. Graph A shows the average FcR values of macrophages recovered from control (line graph ) and BVDV inoculated groups (bar graph). The pre-inoculation values for the in vivo functional assays are represented by day 0. Graph B shows the results of BVDV infection of macrophages in vitro, control (solid line) and BVDV infected (dashed line) which are displayed as the average response of the two calves. Error bars represent the standard deviation values calculated from the means obtained for each animal in a particular group.

centage of AM (70-80%) at days 4 and 6 PI as a result of a slight neutrophil influx. The cell population exposed to BVDV in vitro contained approximately 98% AM and 2% fibroblasts. During the 8 day period of culture there was no substantial decline in the number of macrophages in control or infected cultures, although the number of libroblasts gradually declined to zero after day 4 PI. 3.3. Immunojluorescence Only small numbers (less than 1%) of BVDV positive cells were detected by direct IF on AM recovered from virus inoculated calves between days 4 and 12 PI. Weak diffuse cytoplasmic staining was observed in these cells, but no BVDV positive fluorescence was observed in AM from the uninfected control calves. A smalI number of BVDV positive cells was also detected from day 1 to 4 PI within the cell populations infected with BVDV in vitro. While the staining pattern was typical of BVDV, the intensity of fluorescence was greater than that observed in

202

M.D. Welsh et al. / Veterinary Immunology and Immunopathology 46 (1995) 195-210

DAYS POST-INFECTION

DAYS POST-INFECTION

Fig. 2. The percentage of AM expressing C3R following in vivo (A) and in vitro (B) control or BVDV infection. Graph A shows the average C3R values of macrophages recovered from the control (line graph) and BVDV (bar graph) inoculated groups . Graph B shows the in vitro results, control (solid line) and BVDV infected (dashed line).

the AM population recovered from BVDV infected calves. Positive cells occurred with a frequency of approximately 3% at day 1 PI, declining to less than 0.5Oh by day 3 PI. At no stage were BVDV positive cells detected in lavage populations from the control calves. 3.4. Fe receptor expression Fc receptor (FcR) expression by the AM was severely reduced following in vivo and in vitro BVDV infection (Fig. 1). Only 17% of the macrophages from BVDV infected calves were positive for FcR by day 4 PI, while the pre-inoculation value was 539/o (Fig. 1 (A) ). The reverse effect occurred in the control group which showed a significant (48%) increase by day 4 PI (Fig. 1 (A) ). FcR expression was significantly reduced on AM from all BVDV infected calves from days 4 to 9 PI, with recovery up to and beyond pre-inoculation levels occurring by days 12and 13PI. Macrophages infected in vitro with BVDV (Fig. 1 (B) ) exhibited an initial reduction in FcR expression to 28% by day 1 PI (control value 56%) which lasted until day 2 PI before recovering to reach the control level of expression by day 6

M.D. Welsh et al. / Veterinary Immunology and lmmunopathology

50

0

2

4

5

6

9

46 (1995) 195-210

203

11 12 13

DAYS POST-INFECTION

30L1 L2

3

4

6

a

!

DAYS POST-INFECTION

Fig. 3. The percentage of AM capable of phagocytosis following in vivo (A) and in vitro (B) control or BVDV infection. Graph A displays the results from control (line graph) and BVDV (bar graph) inoculated groups of calves. Graph B shows the in vitro results, control (solid line) and BVDV infected (dashed line).

PI. Significant reductions PI.

in FcR expression

were observed from day 1 to day 4

3.5. Complement receptor expression Fig. 2 shows the effects of BVDV infection on C3R expression. Following in vivo BVDV infection significant reductions in C3R occurred, mainly from day 4 to 9 PI (Fig. 2 (A) ). The greatest impairment in expression occurred at day 6 post virus infection, representing a 7 1% reduction from the pre-infection value. Recovery of C3R to pre-inoculation levels did not occur until day 11 and then exceeded the control value by day 13 PI. During the same period significant increases were observed in C3R expression by macrophages from the control calves (Fig. 2(A)). In vitro BVDV infection of the AM led to a significant reduction in the number of cells expressing C3R between days 3 and 8 PI (Fig. 2 (B) ). By day 3 PI, only 9% of the virus inoculated AM expressed C3R, compared with the control value of 50%. Receptor expression began to recover after day 4 PI and reached a peak of 47% at day 6 PI, which was still considerably lower than the control level of

204

M.D. Welsh et al. / Veterinary Immunology and Immunopathology

46 (1995) 195-210

(A) filO0 5

90

5

00

3

70

I 60

0

2

4

5

6

9

11 12 13

DAYS POST-INFECTION

(6)

pJ

E 4

90

g L-l

2

80

x

70

123466 DAYS POST-INFECTION

Fig. 4. The percentage of AM with microbicidal capacity following in vivo (A) and in vitro (B) control or BVDV infection. Graph A displays the results from control (line graph) and BVDV (bar graph) inoculated groups of calves Graph B shows the in vitro results, control (solid line) and BVDV infected (dashed line).

75%. Although virus inoculated cultures showed a partial recovery in C3R expression, the control level was not attained by the end of the experiment. 3.6. Phagocytosis Prior to BVDV inoculation the mean control level of phagocytosis displayed by the AM was 79% and increased slightly by day 2 PI, before dropping to the lowest value of 60% on day 4 PI (Fig. 3(A) ). Macrophages from the control calves also displayed an increase from the pre-inoculation (day 0) value (Fig. 3 (A) ), but unlike the BVDV inoculated calves the elevated phagocytic activity remained high. Reductions in the level of phagocytosis varied with individual animals in the BVDV challenged groups; however, an overall reduction in the percentage of cells capable of phagocytosis was observed between days 2 and 11 PI, although only four calves showed significant reductions. With cultures infected in vitro, phagocytic ability was severely affected particularly between days 1 and 3 PI (Fig. 3 (B ) ), during which time significant reductions were observed. The largest difference between control and BVDV infected cultures was on day 2 PI, when the values were 88% and 58% respectively, and this was followed by recovery to control levels by day 4 PI.

M.D. Welsh et al. / Veterinary Immunology and

3.7. Microbicidal

Immunopathology

46 (1995) 195-210

205

activity

The level of microbicidal activity of AM, from the control and virus groups of calves, in the pre-inoculation tests was 100%. Following BVDV infection this level fell by 33% after 6 days (Fig. 4 (A) ). Significant reductions in killing ability were observed in seven of the BVDV inoculated calves from day 5 to 11 PI, with recovery apparent from day 9 PI. In cultures infected in vitro no significant difference in microbicidal activity was observed between control and BVDV inoculated macrophages (Fig. 4 (B ) ) throughout the experiment. 3.8. Neutrophil chemotactic factor production NCF production was expressed as the percentage change (increase or decrease) with reference to the pre-inoculation chemotaxis level (Fig. 5 (A ) ). NCF production increased from the pre-inoculation level in the control and BVDV

0

2

4

5

6

9

11 12 13

DAYS POST-INFECTION

1

2

3

4

6

8

DAYS POST-INFECTION

Fig. 5. NCF production by AM following in vivo (A) and in vitro (B) control or BVDV infection. Graph A displays the results from control (line graph) and BVDV (bar graph) inoculated groups of calves. NCF graphs represent the percentage change in NCF production compared with the pre-inoculation values in vivo (graph A) or compared with the uninfected control values in vitro (graph B). Production of NCF by stimulated macrophages cultures was assessed by the ability of supernates to induce a chemotactic response by neutrophils isolated from peripheral blood of uninfected (normal) calves.

206

M.D. Welsh et al. / Veterinary Immunology and Immunopathology

46 (1995) 195-210

inoculated groups; however, this increase was short lived in the virus infected calves (Fig. 5 (A) ), and by day 5 post virus infection a 60% decrease from the pre-inoculation levels was noted. A reduced level of NCF production persisted until at least day 13 PI. All virus-inoculated calves displayed a significant reduction in the amount of NCF being produced at some stage from day 5 PI onwards. NCF production by AM inoculated with BVDV in vitro was expressed as the percentage change (increase or decrease) with reference to the control inoculated cultures (Fig. 5 (B ) ). A reduction of 11% in NCF production was recorded by day 3 PI (BVDV infected cultures); however, this was not significant compared with the controls and no sign&ant differences were recorded throughout the experiment.

4. Discussion One of the many clinical manifestations of BVDV infection is a transient immunosuppression which involves a leukopenia along with direct infection of lymphocytes and macrophages leading to impairment of their normal functions (Howard, 1990). This transient immunosuppression alone may directly lead to predisposition to respiratory disease, however the effects of BVDV infection on AM functions, which could significantly favour the occurrence of disease, have not been investigated in detail. AM are considered to be important in the defence of the lung against invasion by pathogens and any interference with their normal function is likely to seriously compromise local immune defences in the respiratory system. As well as being considered primary respiratory pathogens PI-3 virus, bovine herpesvirus type- 1 (BHV- 1) and RSV have all been implicated as predisposing agents in acute undifferentiated bovine respiratory disease ( Andrews, 1983; Radostits and Townsend, 1989). These viruses have also been reported to interfere with normal AM functions (Brown and Ananaba, 1988; Adair and McNulty, 1992 ) and in so doing are thought to contribute to invasion of the lung by secondary bacterial pathogens (Babiuk et al., 1988). BVDV has frequently been recovered from outbreaks of pneumonia in calves (Bryson, 1985) and is considered by some to be an important predisposing agent in the pathogenesis of bovine respiratory disease. However experimental reproduction of respiratory disease by inoculation of BVDV into calves has produced conflicting results (Radostits and Townsend, 1989). In the present study a BVDV isolate made during a routine diagnostic investigation from a calf with severe pneumonia was used. The aim was to elucidate the effect of the virus on AM functions and help clarify the role of the virus in calf respiratory disease. Neutralising antibodies to BVDV increased by over four-fold in all the inoculated animals, indicating that the calves became infected; however, significant clinical disease was not observed, suggesting that this virus on its own was not capable of inducing significant lung damage. No substantial changes were noted between the percentage of AM recovered by lavage before and after BVDV infection but in general more than 80% of the cells

M.D. Welsh et al. / Veterinary Immunology and Immunopathology 46 (1995) 19.5210

207

had the morphology of macrophages. Lopez et al. ( 1986) reported that inoculation of calves with BVDV did not significantly alter the PMN/AM ratio whereas infection of calves with BHV- 1 or PI-3 virus has been reported to result in significant changes in the lavage cell population (Brown and Ananaba, 1988 ) . Following BVDV infection of the calves, only a small proportion of AM stained positive for BVDV antigen by IF and these cells generally had a weak diffuse .cytoplasmic staining pattern, similar to that described by Toth and Hesse ( 1983). In vitro infection of the AM also yielded only a low percentage of BVDV IF positive cells. This suggested that the cellular dysfunctions which result from BVDV infection may not be dependent on direct cell infection. The in vitro results may suggest the establishment of an anti viral state in the macrophages, as indicated by the lack of widespread infection occurring in the AM population after 8 days in culture. Alternatively the findings may indicate that this isolate of BVDV cannot readily infect AM and relies on other mechanisms to disrupt macrophage functions. However, successful infection appeared to occur in some cells, perhaps suggesting that the stage of macrophage maturation, differentiation or activation is important for BVDV infection and replication, as appears to be the case with Maedi Visna virus, human RSV, and PI-3 virus infection of monocytes/macrophages (Gendelman et al., 1986; Krilov et al., 1987). In a subsequent experiment a higher proportion of AM were infected with this isolate of BVDV under roller culture conditions which tends to suggest that the state of activation of the AM may be important for BVDV infection (results not presented). The conditions required for BVDV infection of AM warrant further study. Although widespread infection of the macrophages was not observed under these culture conditions, substantial functional defects were recorded following BVDV infection. Indirect effects of viruses on AM functions have been noted during other virus infections, e.g. influenza and bovine RSV (Nugent and Pesanti, 1979; Adair and McNulty, 1992; Trigo et al., 1985). Impairment of AM, Fc and complement receptor expression is likely to reduce the recognition of antibody and complement opsonised pathogens and may also interfere with antibody-dependent cellular cytotoxic (Forman and Babiuk, 1982)) and complement-dependent cytotoxic mechanisms (Grewal and Babiuk, 1980; Rouse et al., 1977 ). Reduction in phagocytosis of complement opsonised C. krusei cells cannot be explained completely by reduction in C3R expression, but rather indicates interference with the range of phagocytic pathways. However, even if invading pathogens are engulfed through phagocytosis, the in vivo results presented here indicate that interference by BVDV with microbicidal pathways may prevent their destruction. AM (amongst other cells) are known to produce leukotriene B4 (LTB4) (Fels et al., 1982) which has potent chemotactic properties (Pettipher et al., 1992 ). In vivo BVDV infection resulted in impaired NCF production which could be interpreted as an inhibition in LTB4 synthesis which has been demonstrated following in vitro BVDV infection of bovine mononuclear cells (Atluru et al., 1992). Depressed NCF production by AM has also been reported following BHV-1 virus infection ( McGuire and Babiuk, 1984). Neutrophils have important anti-viral

208

M.D. Welsh et al. / Veterinary Immunology and Immunopathology 46 (I 995) 195-210

roles (Grewal et al., 1977; Grewal and Babiuk, 1980), and any reduction in their recruitment to sites of infection may severely weaken lung defences. BVDV has also been shown to impair neutrophil function, which is likely to further exacerbate the problem (Roth et al., 198 1). No significant decreases in killing ability and NCF production were noted in cultures infected in vitro. The differences between the results with these tests in vivo and in vitro, and the possible biphasic phagocytic response observed with macrophages from BVDV infected calves lavaged on days 4 and 9 PI (Fig. 3 (A ) ) , may indicate that immune mechanisms are involved in AM dysfunction. A similar phenomenon of immune mediated cellular dysfunction has been observed following influenza virus infection (Jakab and Warr, 1983) and biphasic fever and leukopenia have been observed during acute BVDV infections (Traven et al., 1991). Although the inoculated calves had BVDV-specific maternal antibodies, the virus was still capable of inducing AM dysfunction. The presence of maternal antibodies may have limited the severity of the infection, however all calves showed a significant rises in BVDV antibody titres Menanteau-Horta et al. ( 1985) also demonstrated seroconversion following administration of a modified-live BVDV vaccine, in the presence of virus-specific maternal antibodies. Also, Howard et al. ( 1989) reported that passively acquired antibody was effective in reducing the extent of respiratory infection but did not make the animals totally refractile to BVDV infection. The results demonstrate that BVDV can severely affect normal AM functions following infection. The cellular defects observed in the AM from BVDV infected calves are likely to be controlled by factors which disrupt other cellular functions during acute BVDV infection (i.e. cytokine imbalance or the release of other cellular metabolites). These factors may not be present in the in vitro situation (owing to absence of lymphocytes), hence the observed differences from the in vivo results. It has been reported that BVDV can cause the release of immunosuppressive substances from BVDV infected cell cultures (possibly prostaglandins) (Markham and Ramnaraine, 1985). We have demonstrated that this isolate of BVDV is capable of inducing elevated levels of prostaglandin E-2 (PGE-2) synthesis from infected FCL cells in culture, to levels possibly considered suppressive to Tcell mitogenesis (M.D. Welsh and B.M. Adair, unpublished data, 1994). The release of PGE-2 by infected cells in the upper respiratory tract could cause a localised immunosuppression (without systemic immunosuppression and infection of lymphocytes) which may be sufficient to induce respiratory disease and promote secondary bacterial infections. This may indicate a novel mechanism for BVDV induced respiratory disease. Release of prostaglandins by BVDV infected cells and alteration of normal cytokine pathways, which may occur by infection of lymphocytes, could also explain the indirect disruption of normal AM functions which have been observed in these studies. Further investigations into the mechanisms which underlie the effects described here are underway and will be reported in due course.

M.D. Welsh et al. / Veterinary Immunology and fmmunopat~o/o~ 46 (I 995) 195-210

209

Acknowledgement

This work was funded by The Department of Agriculture for Northern Ireland.

References Adair, B.M. and McNulty, M.S., 1992. Effect of in vitro exposure of bovine alveolar macrophages to different strains of bovine respiratory syncytial virus. Vet. Immunol.Immunopathol., 30: 193206. Andrews, A.H., 1983. Respiratory disease in calves. Vet. Annu., 23: 48-55. Atluru, D., Gudapaty, S., Xue, X., Gurria, F., Chengappa, M.M., McVey, D.S., Minocha, H.C. and Atluru, S., 1992. In vitro inhibition of 5-lipoxygenase metabolite, leukotriene B4, in bovine mononuclear cells by bovine viral diarrhoea virus. Vet. Immunol. Immunopathol., 3 1: 49-59. Babiuk, L.A., Lawmann, M.J.P. and Bielefeldt Ohmann, H., 1988. Viral-bacterial synergistic interaction in respiratory disease. Adv. Virus Res., 35: 2 19-249. Barker, J.C., 1990. Clinical aspects of bovine virus diarrhoea virus infection. Rev. Sci. Tech. Off. Int. Epiz.,9(1):25-41. Bishop. O.N., I98 I. Statistics for Biology, 3rd edn. Longman, London. Brown, T.T. and Ananaba, G., 1988. Effect of respiratory infections caused by bovine herpesvirus- I or parainfluenza-3 virus on bovine alveolar macrophage functions. Am. J. Vet. Res., 49: 14471451. Bryson, D.G., 1985. Calfpneumonia. Vet. Clin. North Am. Food Anim. Pratt., I: 237-257. Fels, A.O.S. and Cohn, Z.A., 1986. The alveolar macrophage. J. Appl. Physiol., 60: 353-369. Fels, A.O.S., Pawlowski, N.A., Cramer, E.B., King, T.K.C., Cohn, Z.A. and Smith, W.A., 1982. Human alveolar macrophages produce leukotriene B4. Proc. Natl. Acad. Sci. USA, 79:7866-7870. Forman, A.J. and Babiuk, L.A., 1982. Effect of infectious bovine rhinotracheitis virus infection on bovine alveolar macrophage function. Infect. Immun., 35: 104 I- 1047. Gendelman, H.E., Narayan, O., Kennedy-Stoskopf, S., Kennedy, P.G.E., Ghotbi, Z., Clements, J.E.. Stanley, J. and Pezeshkpour, G., 1986. Tropism of sheep lentiviruses for monocytes: Susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages. J. Virol., 58: 67-74. Grewal, A.S. and Babiuk, L.A., 1980. Complement-dependent. polymorphonuclear neutrophil-mediated cytotoxicity of herpesvirus-infected cells: Possible mechanism(s) of cytotoxicity. Immunology,40: 151-161. Grewal, A.S., Rouse, B.T. and Babiuk, L.A.. 1977. Mechanisms of resistance to herpesviruses: Comparison of the effectiveness of different cell types in mediating antibody-dependent cell-mediated cytotoxicity. Infect. Immun., 15: 698-703. Howard, C.J., 1990. Immunological responses lo bovine virus diarrhoea virus infections. Rev. Sci. Tech. Off. Int. Epiz., 9( 1): 95-103. Howard, C.J., Clarke, M.C. and Brownlie, J., 1989. Protection against respiratory infection with bovine virus diarrhoea virus by passively acquired antibody. Vet. Microbial., 19: 195-203. Jakab, G.T. and Warr, G.A., 1983. The participation of antiviral immune mechanisms in alveolar macrophage dysfunction during viral pneumonia. Bull. Europ. Physiopathol. Resp.. 19: 173-l 78. Khadom, N.J., Dedieu, J.F. and Viso, M., 1985. Bovine alveolar macrophage: A review. Ann. Rech. Vet., 16: 175-183. Krilov, L.R., Hendry, R.M., Godfrey, E. and McIntosh, K., 1987. Respiratory virus infection of peripheral blood monocytes: Correlation with ageing of cells and interferon production in vitro. J. Gen. Viral., 68: 1749-1753. Lopez, A., Maxie, M.G., Ruhnke, L., Savan, M. and Thomson, R.G., 1986. Cellular inflammatory response in the lungs of calves exposed to bovine viral diarrhea virus. Am. J. Vet. Res.. 47: l2831286.

210

M.D. Welsh et al. / VeterinaryImmunology andlmmunopathology 46 (1995) 195210

Markham, R.J.F. and Ramnaraine, M.L., 1985. Release of immunosuppressive substances from tissue culture cells infected with bovine viral diarrhea virus. Am. J. Vet. Res., 46: 879-883. McGuire, R.L. and Babiuk, L.A., 1984. Evidence for defective neutrophil function in lungs of calves exposed to infectious bovine rhinotracheitis virus. Vet. Immunol. Immunopathol., 5: 259-271. McNulty, MS. and Allan, G.M., 1984. Applications of immunofluorescence in veterinary viral diagnosis. In: M.S. McNulty and J.B. McFerran (Editors), Recent Advances in Virus Diagnosis. Martinus Nijhoff, The Hague, pp. 15-26. Menanteau-Horta, A.M., Ames, T.R., Johnson, D.W. and Meiske, J.C., 1985. Effect of maternal antibody upon vaccination with infectious bovine rhinotracheitis and bovine virus diarrhea vaccines. Can. J. Comp. Med., 49: IO- 14. Morahan, P.S., Connor, J.R. and Leary, K.R., 1985. Viruses and the versatile macrophage. Br. Med. Bull., 41: 15-21. Nugent, K.M. and Pesanti, E.L., 1979. Effect of influenza infection on the phagocytic and bactericidal activities of pulmonary macrophages. Infect. Immun., 26: 65 l-657. Pettipher, E.R., Higgs, GA. and Salmon, J.A., 1992. Eicosanoids (prostaglandins and leukotrienes). In: J.T. Whither and S.V. Evans (Editors), Biochemistry of Inflammation. KIuwer Academic. London, pp. 91-109. Potgieter, L.N.D., 1988. Immunosuppression in cattle as a result of bovine viral diarrhea virus infection. Agri-Practice, 9: 7-14. Radostits, O.M. and Townsend, H.G., 1989. The controversy surrounding the role of bovine virus diarrhea virus (BVDV) in the pathogenesis of pneumonic pasteurellosis in cattle. Proc. Am. Assoc. Bov. Pratt., 2 I: I 1 1-I 14. Reggiardo, C., 1979. Role of BVD virus in shipping fever of feedlot cattle. Case studies and diagnostic considerations. American Association of Veterinary Laboratory Diagnosticians, 22: 3 15-320. Roth, J.A., Kaeberle, M.L. and Griffith, R.W., I98 I. Effects of bovine viral diarrhoea virus infection on bovine polymorphonuclear leukocyte function. Am. J. Vet. Res., 42: 244-250. Rouse, B.T., Grewal, AS. and Babiuk, L.A., 1977. Complement enhances antiviral antibody-dependent cell cytotoxicity. Nature, 266: 456-458. Toth, T.H. and Hesse, R.A., 1983. Replication of five bovine viruses in cultured bovine alveolar macrophages. Arch. Viral., 75: 2 19-224. Traven, M., Alenius, S., Fossum, C. and Larsson, B., 1991. Primary bovine viral diarrhoea virus infection in calves following direct contact with a persistently viraemic calf. J. Vet. Med. B, 38: 453-462. Trigo, E., Liggitt, H.D., Evermann, J.F., Breeze, R.G., Houston, L.Y. and Silflow, R., 1985. Effect of in vitro inoculation of bovine respiratory syncytial virus on bovine pulmonary alveolar macrophage function. Am. J. Vet. Res., 46: 1098-l 103.