Modulation of bovine neutrophil antibacterial activities by Pasteurella haemolytica A1 purified capsular polysaccharide

Microbial Pathogenesis 1989; 6 : 133-141

Modulation of bovine neutrophil antibacterial activities by Pasteurella haemolytica Al purified capsular polysaccharide Charles J . Czuprynski,' E . J . Noel' and C . Adlam 2 'Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wl, U.S.A . and 2Department of Bacteriology, The Wellcome Research Laboratories, Beckenham, Kent, U .K. (Received September 9 1988; accepted in revised form December 8, 1988)

Czuprynski, C. J . (Dept. of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI, U .S .A .), E . J . Noel and C. Adlam . Modulation of bovine neutrophil antibacterial activities by Pasteurella haemolytica A1 purified capsular polysaccharide . Microbial Pathogenesis 1989 ; 6 : 133-141 . Preincubation of bovine neutrophils with Pasteurella haemolytica Al purified capsular polysaccharide markedly diminished their ability to ingest and kill P. haemolytica, but not Escherichia coli, in vitro . Ingestion and killing were impaired even when the P. haemolytica were preopsonized, thus suggesting that the inhibitory effects of the polysaccharide included a direct effect on bovine neutrophils rather than simply competition for serum opsonins . Preincubation of neutrophils with purified polysaccharide did not elicit a chemiluminescence response, nor did it alter the chemiluminescence response of neutrophils to subsequent stimulation with opsonized P. haemolytica or opsonized zymosan. In addition, purified polysaccharide alone was neither chemotactic nor did it induce a shape change in bovine neutrophils . These data suggest that the deposition of capsular polysaccharide in the lung during the onset of pulmonary pasteurellosis might impair the ability of neutrophils to ingest and kill P . haemolytica . The capsular polysaccharide of P. haemolytica, therefore, may contribute in part to the fibrinous pleuropneumonia that characterizes acute pasteurellosis . Key words : Pasteurella haemolytica ; capsule; polysaccharide ; neutrophils ; bovine ; antibacterial .

Introduction

Pasteurella haemolytica Al is an important component of the bovine respiratory disease complex (BRD) that is the principal source of economic loss for feedlot cattle in North America' and is of considerable importance to the cattle and sheep industries in other countries as wel1 . 2 P. haemolytica Al causes an acute fibrinous pleuropneumonia, particularly in animals concurrently infected with bovine herpesvirus-1 or some other viral respiratory pathogen .' P. haemolytica Al possesses at least two virulence determinants that are thought to contribute to the pathogenesis of pulmonary pasteurellosis . The first is an extracellular cytotoxin that can impair various functions of leukocytes and other cells . 3 The second is a polysaccharide capsule that is present on the surface of some strains of P. haemolytica Al during log-phase *Author to whom correspondence should be addressed : Dr C. J . Czuprynski, Department of Pathobiological Sciences, School of Veterinary Medicine, 2015 Linden Drive West, Madison, WI 53706, U .S .A . 0882-4010/89/020133+09$03 .00/0

© 1989 Academic Press Limited



134

Charles J . Czuprynski et a/.

growth . 4-' This capsular polysaccharide, which is rather loosely associated with the bacterial surface,$ has been purified and its chemical structure elucidated as -3)ManNAc-(f31-4)-Man NAcA-(fll- . 9 Although there are no previous reports of how this purified capsular polysaccharide might influence the host response to P. haemolytica, immunization with crude salicylate extracts of P. haemolytica, that likely contain substantial amounts of capsular polysaccharide, have been shown to confer some protection against experimental pasteurellosis . ) 1011 In addition, an association between the presence of pre-existing circulating antibodies against the antigens contained within saline extracts of P . haemolytica Al and resistance to pasteurellosis in cattle has been reported by other investigators ." During pulmonary pasteurellosis, neutrophils are a prominent cell type in the lung where they may contribute both to host defense and to the extensive tissue damage and loss of pulmonary function that occurs during the acute disease .""' We have reported previously that bovine blood neutrophils are capable of ingesting and killing some strains of P. haemolytica Al in vitro ." In the present study we determined how the presence of free capsular polysaccharide might alter the ability of bovine neutrophils to ingest, and kill P . haemolytica Al in vitro .

Results Effects of capsular polysaccharide on ingestion of P . haemolytica A 1 by bovine neutrophils Preincubation of bovine neutrophils with purified capsular polysaccharide markedly impaired their ability to ingest an unencapsulated strain of P . haemolytica Al (Fig . 1 ) . Significant inhibition (P < 0 .05) of both the percentage of neutrophils that ingested P. haemolytica [Fig . 1 (a) and the phagocytic index [Fig . 1 (c)] was seen with as little as 0 .1 µg polysaccharide, whereas significant inhibition (P < 0 .05) of the mean number of bacteria per ingestory neutrophil occurred in the presence of 1 mg or more polysaccharide [Fig . 1 (b)] . Maximal inhibition of neutrophil ingestion of P . haemolytica was observed at concentrations of 10-100 µg capsular polysaccharide . Although competition for antibodies against P . haemolytica Al might be one mechanism by which ingestion was diminished, significant inhibition of ingestion was observed when preopsonized P. haemolytica Al were used (Fig . 2), thus suggesting that the capsular polysaccharide may have some direct effect on the ability of bovine neutrophils to ingest P. haemolytica Al . In contrast, neutrophils preincubated with 10 ,ug purified polysaccharide were unimpaired in their ability to ingest Escherichia coli as compared to control neutrophils (Fig . 1 ) . Effects of capsular polysaccharide on killing of P . haemolytica A 1 by bovine neutrophils Preincubation of bovine neutrophils with purified capsular polysaccharide resulted in a dramatic reduction in their ability to kill P. haemolytica Al (Fig . 3) . Neutrophil killing of P. haemolytica Al was slightly reduced (P < 0 .19) at 0 .1 µg, nearly nonexistent at 1 .0 µg (P < 0 .05), and completely ablated at 10 /tg (growth of P. haemolytica Al equivalent to that in neutrophil-free control tubes was observed) . In contrast, neutrophil killing of E. coli was unimpaired by preincubation with 10 yg capsular polysaccharide (Fig . 3) . Effects of capsular polysaccharide on bovine neutrophil luminol-enhanced chemiluminescence Release of reactive oxygen intermediates from stimulated neutrophils is thought to be a principal mechanism by which they exert antibacterial activity and indiscriminately



P. haemolytica polysaccharide and bovine PMNS

100

135

a

W

0

40

0 . c

u a

40

0

r

6 .0

0. 0

3

5 .0

a

4 .0

m c

0

3 .0 0

a

m a E

c m

2 .0 1 .0

0

0 .0

500 C

400 x a' v c

i

300 V rn O G

200

o.

100

s I 1 I l I I t 1 .0 10 100 0 0 .001 0.01 0 .1 Capsular polysaccharide (pg/ml )

Fig . 1 . Preincubation of bovine neutrophils with P. haemolytica capsular polysaccharide markedly impairs their ability to ingest P. haemolytica in vitro (0) . Results are expressed as the mean + SEM (six separate experiments) percentage of neutrophils ingesting bacteria (a), the mean number of bacteria per ingesting neutrophil (b) and the phagocytic index (c, see Materials and methods section) . Asterisks indicate P < 0 .05 as compared to control neutrophils . Ingestion of E. coli by control neutrophils and by neutrophils incubated with 10 µg polysaccharide are indicated by (0) (mean ± of SEM of five separate experiments) .

cause local tissue damage . We, therefore, decided to examine the influence of capsular polysaccharide on the ability of neutrophils to release oxygen intermediates, as determined by their chemiluminescence activity . Incubation of neutrophils with 0 .1100 pg polysaccharide alone did not directly stimulate a chemiluminescence response



Charles J . Czuprynski et al.

136 350 300 , 250 v v c 200

100 50 0 0

0 .01 0 .1 1 .0 Capsular polysaccharide (µg/ml)

10

Fig . 2 . Preopsonization of P. haemolytica with pooled bovine sera does not prevent polysaccharide mediated inhibition of phagocytosis . Results are expressed as the mean + SEM (two separate experiments) phagocytic index .

(Fig . 4), nor did preincubation with these concentrations of polysaccharide have any effect on the chemiluminescence response of neutrophils subsequently stimulated with opsonized P. haemolytica Al or opsonized zymosan (Fig . 4) . Neutrophil migration and shape change Purified polysaccharide by itself was not chemotactic for bovine neutrophils (data not shown) . In addition, preincubation of neutrophils with 10 µg purified polysaccharide alone elicited a significant shape change in only 4% of the neutrophils, whereas incubation in zymosan-activated bovine serum elicited a dramatic shape change in 85% of the neutrophils from the same donor . Although preincubation with purified polysaccharide enhanced the migration of neutrophils from some donors towards

3.0 2 .5 2 .0 0 c

1 .5

0

1 .0

0 _j

0 .5 0.0 -0 .5 -1 .0

0

0 .001

0 .01 0 .1 Capsular polysaccharide

1 .0

10 .0

(µg/ml)

Fig . 3 . Killing of P. haemolytica by bovine neutrophils is progressively decreased by preincubation with increasing concentrations of capsular polysaccharide (0) . Results are expressed as the mean±SEM (seven separate experiments) log10 reduction in P. haemolytica during a 3 h incubation in vitro . In the absence of neutrophils P. haemolytica increased in number (A) during this incubation period . Asterisks indicate P < 0.05 as compared to control neutrophils . Killing of E. co/i by control neutrophils and neutrophils preincubated with 10 pg polysaccharide are indicated by (0) (mean±SEM of five separate experiments) .



P. haemolytica polysaccharide and bovine PMNS

1 37

7.0

6 .0

3 .0

2 .0

O i

0

O

I

0.1

-

-0

I

i

1 .0

10

Capsular polysaccharide (µg/ml) Fig . 4 . P. haemolytica capsular polysaccharide does not elicit chemiluminescence (0) by bovine neutrophils nor alter their chemiluminescence response to opsonized P . haemolytica (0) or opsonized zymosan (/) . Results are expressed as the mean + SEM (four-five separate experiments) log lo CPS of the peak chemiluminescence response. Capsular polysaccharide also had no effect on the kinetics of the chemiluminescence response . (Data not shown) .

zymosan-activated serum, this response was not consistently observed for neutrophils from all blood donors (data not shown) . Discussion The results of this study indicate that P. haemolytica Al purified capsular polysaccharide modulates the ability of neutrophils to ingest and kill an unencapsulated strain of P . haemolytica Al in vitro . Because the purified capsular polysaccharide contains less than 1% protein, it is unlikely that the previously described outer membrane proteins of P. haemolytica 16 contributed significantly to the observed functional alterations of bovine neutrophils . These observations also are unlikely to be influenced significantly by contaminating lipopolysaccharide (LPS) or cytotoxin . The purified polysaccharide used in this study has been described biochemically in great details and is substantially free of LPS as determined by its low reactivity in the limulus amoebocyte lysate assay (we calculate that 1 .0 µg purified polysaccharide has approximately 5 .0 pg LPS), its low pyrogenicity for rabbits, and its sugar constituents .' These low estimated levels of LPS in the limulus assay may not be due to contaminating LPS but rather to the highly charged nature of the purified polysaccharide ." The effects of purified capsular polysaccharide on bovine neutrophils also differ considerably from those reported for neutrophils treated with phenol-water extracted LPS from P . haemolytica .' $ In addition, we find the purified capsular polysaccharide to be completely free of cytotoxic activity against bovine neutrophils at concentrations of up to 0 .5 mg per ml (data not shown) as assessed by a previously described colorimetric assay for quantitating the lethal effects of P. haemolytica cytotoxin .' 9,20 Preincubation of bovine neutrophils with purified polysaccharide dramatically decreased their ability to ingest (Fig . 1) and kill (Fig . 3) P. haemolytica Al . Although this might simply reflect competition of the polysaccharide for opsonizing antibodies present in bovine sera, a substantial impairment of neutrophil ingestion of P . haemolytica was observed even when the bacteria were preopsonized with bovine sera before they were added to polysaccharide-treated neutrophils (Fig . 2) . Some



138

Charles J . Czuprynski et al.

specificity of the polysaccharide-mediated suppression of phagocytosis and killing must occur, however, because polysaccharide-treated bovine neutrophils were unimpaired in their ability to ingest and kill E. coli (Figs 1 and 3) . In a previous study it was reported that incubation with mannose markedly reduced the oxidative response of human neutrophils to various stimuli, presumably via bonding to mannose receptors on the neutrophil surface ." Because the P . haemolytica capsular polysaccharide has a ManNAc-backbone, one might postulate a similar mechanism for the inhibition of bovine neutrophil antibacterial activity . Unlike mannose-treated human neutrophils, however, polysaccharide-treated bovine neutrophils were unaffected in their ability to undergo a chemiluminescence response to opsonized P. haemolytica and opsonized zymosan . These data suggest that the initial interaction between polysaccharide-treated neutrophils and P. haemolytica was sufficient to elicit a vigorous oxidative burst, but subsequent internalization and killing of the bacteria were blocked . The biological effects of bacterial capsules are often thought of as restricted principally to the physical barrier they present to effective opsonization and phagocytosis of encapsulated bacteria . Other investigators have reported, however, that bacterial capsular polysaccharides may influence neutrophil function in a manner similar to what we report here . For example, the capsular polysaccharide of Bacteroides fragilis is capable of inducing abscess formation in vivo 22 and blocking the bactericidal activity of human neutrophils in vitro . 23 Light and electron microscopic studies have clearly demonstrated the presence of a polysaccharide layer on the surface of certain strains of log-phase P. haemolytica Al . 4-9 The serotype-specific antigen of P . haemolytica Al, which has been cloned into E. co/i, contains considerable polysaccharide and may represent some form of the capsular polysaccharide . 24 Encapsulation is not lost after repeated passage of P . haemolytica in vitro, 25 and is most prominent on bacteria in the logarithmic phase of growth . 5-7 The capsular polysaccharide apparently is rather loosely bound to the bacterial surface and can be removed by gentle incubation with sodium chloride, sodium salicylate, or potassium salicyclate . 8 '9 .26 .2 ' These crude extracts, which are mixtures of capsular polysaccharide and other bacterial components, contain antigens that are recognized by sera 12,2' and lung lavage fluid 28 from calves vaccinated with a bacteria or infected with live P. haemolytica . Some correlation has been reported between resistance to transthoracic challenge with virulent P. haemolytica Al and serum antibodies directed against these crude extra cts . 12 Immunization of cattle,"' sheep, 11 and laboratory rodents 10 with these crude extracts imparts some resistance to subsequent challenge with P. haemolytica . Although the specific component in these crude extracts that is responsible for these responses has not been elucidated, it is reasonable to propose that capsular polysaccharide is a likely candidate . In summary, these data indicate that the capsular polysaccharide of P . haemolytica Al can significantly modulate bovine neutrophil antibacterial activity . In vivo production of capsular polysaccharide by P. haemolytica multiplying within the lungs of infected calves, and deposition of this material within the alveoli, has been reported . 29 Taken as a whole these results suggest that the ability of neutrophils to ingest and kill P . haemolytica multiplying in the lung might be impaired, at least in part, by the presence of capsular polysaccharide . The detrimental effects of capsular polysaccharide, along with other components such as P. haemolytica cytotoxin and LPS, may contribute to the acute pleuropneumonia that is characteristic of pulmonary pasteurellosis . Materials and methods

Bacteria . A lyophilized culture of P . haemolytica Al was obtained from Dr G . Frank (National Animal Disease Center, Ames, IA) . We have reported previously on the ingestion and killing of



P. haemolytica polysaccharide and bovine PMNS

139

this strain, which has little encapsulation and produces low amounts of cytotoxin, by bovine neutrophils in vitro, 16 •20 The culture was reconstituted in brain-heart infusion broth (Difco, Detroit, MI) checked for purity by plating on blood agar, and then used to inoculate fresh broth that was incubated overnight at 37°C . Glycerol (15% final concentration) was added as a cryo preservative and the culture frozen as aliquots at -70°C. Before each experiment an aliquot would be removed and the frozen surfaces scraped with a sterile loop . This was streaked on blood agar and incubated at 37°C for 18 h . The bacteria were washed from the surface with sterile saline, washed several times in PBS, and resuspended in Hanks' balanced salts solution that contained 0 .25% bovine serum albumin (HBSA) . A different isolate of P. haemolytica Al obtained from Dr R . Corstvet, Baton Rouge, LA), which produces copious amounts of cytotoxin, was used for the production of cytotoxin and maintained as described above . Escherichia coli ATCC 25922 was obtained from the clinical microbiology laboratory of the University of Wisconsin Veterinary Medicine Teaching Hospital and maintained as described above. P . haemolytica A 1 purified capsular polysaccharide . Capsular polysaccharide was purified from the culture supernatant of a broth culture of P. haemolytica Al as described previously in detail .' Preparation of blood neutrophils and preincubation with purified capsular polysaccharide . Blood was collected from the jugular vein of healthy adult mixed-breed (predominantly Holstein) cattle using 0 .38% sodium citrate (final concentration) as the anticoagulant . The blood was centrifuged at 400 xg for 20 min and the buffy-coat layer removed . Erythrocytes were eliminated by hypotonic lysis30 and the leukocytes were resuspended (50x106 cells per ml) in calcium and magnesium-free Hanks' balanced salts solution that contained 0 .1 M EDTA and 0 .25% bovine serum albumin . Two ml of this suspension was layered onto a discontinuous Percoll gradient (2 ml 60% Percoll over 3 ml 68% Percoll) and centrifuged at 400xg for 15 min at room temperature . The cell pellet (usually 95% neutrophils, > 98% viable) was removed, washed several times and resuspended in Hanks' balanced salts solution . Cell number was estimated using a hemacytometer . All cell suspensions and solutions were maintained at room temperature because we find that maintaining bovine neutrophils at 4°C impairs their activity in vitro . The neutrophils were preincubated with graded doses of purified capsular polysaccharide (0 .01100 µg per ml) at a cell concentration of 4x106 per ml in HBSA for 1-2 h at 39°C . Control neutrophils were preincubated in HBSA without capsular polysaccharide . Following this preincubation, which had no adverse effect on neutrophil number or viability, the cells were used in the various in vitro assays . The neutrophils were not washed before being used, therefore, the purified polysaccharide was present throughout all subsequent incubations . Neutrophil shape change . Changes in the morphology of neutrophils after exposure to potential chemoattractants was assessed by a modification of a previously described method ." Neutrophils (2x106 ) were suspended in 0 .5 ml HBSS that contained 0 .1-100 µg purified polysaccharide or 25% (final volume) zymosan activated sera as a positive control . After incubation for 1 h at 37°C, 0 .2 ml was removed from each tube and placed into separate tubes that contained 0 .5 ml cold 2% glutaraldehyde . Samples were then removed from each tube, placed into a hemacytometer, and examined microscopically at 400x magnification . Cells that retained their spherical shape were counted as negative, whereas significant formation of blebs or elongation was considered to be a positive shape change . Microchamber chemotaxis assay. Neutrophil migration was assessed using a 48 well micro chemotaxis chamber (Neuro Probe, Inc ., Cabin John, MD) as described previously ." The lower wells of the chamber were filled with 0 .03 ml of chemoattractant (10-25% zymosan-activated bovine sera in HBSS) ; control wells received warm HBSS . A 3,um pore PVP-free polycarbonate filter (Neuro Probe, c at . n o. NFB 3) was carefully placed shiny side up on top of the filled wells . The chamber then was assembled and the entire unit preincubated at 39°C for 10-15 min . Neutrophils (2x10 ° in 0.05 ml HBSA) were added to each of the upper chambers of the wells and the apparatus placed in a small humidified tray and incubated at 39°C for 20-30 min . After incubation, the chamber was disassembled, and the filter was removed and stained with Diff-Quik ® (American Scientific Products, McGaw Park, IL) . Neutrophil migration through the filter in each well was quantitated by counting the number of neutrophils in five separate highpower (400x magnification) fields . Three to four wells were prepared and examined for each experimental condition . Results were expressed as the mean number of cells per high power field . To normalize the results obtained in separate experiments with different blood donors,



Charles J . Czuprynski et al.

140

the number of cells per high power field for neutrophils preincubated with polysaccharide was expressed as a percentage of the number of neutrophils per high power field for untreated control neutrophils preincubated in HBSA . Control wells that contained HBSS in the lower chamber, rather than zymosan activated sera, typically had no or very few neutrophils that migrated through the filter . Phagocytosis and killing of P . haemolytica . Phagocytosis and killing of P. haemolytica Al by bovine neutrophils was determined as described previously . 15• 20 Briefly, phagocytosis was assessed by incubating 12 .5X106 washed P. haemolytica with 2 .5x 106 neutrophils and 0 .1 ml pooled adult bovine sera in a total volume of 1 .0 ml HBSA . After incubation at 37°C for 30 min (rotation at 8 rpm) the uningested bacteria were removed by three slow-speed centrifugations (100xg for 5 min at 4°C) and the neutrophils resuspended in HBSA . Cytocentrifuge smears were prepared, stained with Diff-Quik', and examined microscopically (1 000x magnification) to determine the percentage of neutrophils that ingested bacteria and the number of bacteria per ingesting neutrophil . Results are also expressed as a phagocytic index calculated as the percentage of neutrophils that contained bacteriaxthe mean number of bactera per ingesting phagocytex100 . In some experiments, P. haemolytica was preopsonized with pooled normal bovine serum (50% final value) for 30 min at 37°C, washed twice with cold HBSA, and used as the target for phagocytosis by bovine neutrophils . Killing of P. haemolytica was determined by incubating 2 .5x10 6 neutrophils and 2 .5x106 washed P. haemolytica Al with 0 .1 ml pooled adult bovine sera in a total volume of 1 .0 ml HBSA at 37°C for 3 h . Samples were removed from tubes before and after incubation, serially diluted in sterile distilled water and plated on blood agar . Colonies were counted after 24 h incubation at 37°C . Results are expressed as the log 10 reduction in viable P. haemolytica as compared to the inoculum . Chemiluminescence. Luminol-enhanced chemiluminescence was determined as described previously .", ", Neutrophils (5xl0 5 ) and luminol (5xl0 -5 M) were placed into polystyrene tubes (#55 .484, Sarstedt Inc ., Princeton, NJ) and preincubated for 15-30 min in a Picolite Luminometer (Packard Instruments Co ., Downers Grove, IL) . Opsonized P. haemolytica (5x10') was injected into the tubes to stimulate chemiluminescence . Baseline unstimulated chemiluminescence was determined for tubes containing neutrophils that received HBSS rather than P. haemolytica . Reaction tubes were counted for 5 s intervals for 35 min at 39°C . The data are expressed as the mean±SEM (triplicate tubes) log 10 cpm of the peak chemiluminescence response for each treatment group . Evaluation of chemiluminescence by integration of the area under the curve as performed by a software package (Cell-Lum, version 2 ; Los Alamos Diagnostic Laboratories, Los Alamos, NM) yielded similar results for each experimental group . Assessment of cytotoxic activity for bovine neutrophils . The cytotoxic activity of purified capsular polysaccharide was assessed using a previously described colorimetric assay ." Culture supernatants from a strain of P. haemolytica Al known to produce large amounts of cytotoxin were prepared and used as a positive control for killing of neutrophils as described previously . 20 Statistical analysis . Data were analyzed for statistical significance by a one-way analysis of variance using the BMDP statistical software package (University of California Press, Berkeley, CA, 1981) . If a significant F value was obtained, then relevant comparisons were made by paired t tests . The levels of significance for all comparisons was set at P < 0 .05 . This work was supported by funds from the United States Department of Agriculture (86CRSR-2-2847 and 87-CRSR-1-2308) . We gratefully acknowledge the efforts of Mr J . M . Knights in the initial purification and characterization of the polysaccharide . We thank the School of Veterinary Medicine Word Processing personnel for the preparation of the manuscript . References 1 . Yates WDG, Stockdale PHG, Babiuk LA, Smith RJ . Prevention of experimental bovine pneumonic Pasteurellosis with an extract of Pasteurella haemolytica . Can J Comp Mad 1983 ; 47 : 250-6 . 2 . Gilmour NJL, Martin WB, Sharp JM, Thompson DA, Wells PW . The development of vaccines against pasteurellosis in sheep . Vet Rec 1979 ; 111 : 15 . 3 . Shewen PE, Wilkie BN . Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes . Infect Immun 1982 ;35 :91-4 . 4.

Beesley JE, 0rpin A, Adlam C . A comparison of immunoferritin, immunoenzyme and gold-labelled



P. haemolytica polysaccharide and bovine PMNS

141

protein A methods for the localization of capsular antigen on frozen thin sections of the bacterium Pasteurella haemolytica. H istochem J 1982; 14: 803-10 . 5. Corstvet RE, Gentry MJ, Newman PR, Rummage JA, Confer AW . Demonstration of age-dependent capsular material on Pasteurella haemolytica serotype Al . J Clin Microbiol 1982 ; 16 :1123-6 . 6. Gilmour NJL, Menzies JD, Donachie W, Frazer J . Electron microscopy of the surface of Pasteurella haemolytica . J Mad Microbiol 1985 ; 19 :25-34 . 7 . Morck DW, Raybould TJG, Acres SD, eta!. Electron microscopic description of glycocalyx and fimbriae on the surface of Pasteurella haemolytica-A1 . Can J Vet Res 1987 ; 51 : 83-8 . 8 . Gentry MJ, Corstvet RE, Panciera RJ . Extraction of capsular material from Pasteurella haemolytica. Am J Vet Res 1982 ; 43 : 2070-3 . 9 . Adlam C, Knights JM, Mugridge JC, eta/. Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica (serotype Al) organisms . J Gen Microbiol 1984 ; 130 : 2415-26 . 10 . Tadayon RA, Lauerman LH . The capacity of various fractions of Pasteurella haemolytica to stimulate protective immunity in mice and hamsters . Vet Microbiol 1981 ; 6 : 85-93 . 11 . Gilmour NJL, Martin WB, Sharp JM, Thompson DA, Wells PW, Donachie W . Experimental immunization of lambs against pneumonic pasteurellosis . Res Vet Sci 1983 ; 35 : 80-6 . 12 . Confer AW, Lessley BA, Panciera RW, Fulton J, Kreps A. Serum antibodies to antigens derived from a saline extract of Pasteurella haemolytica : correlation with resistance to experimental bovine pneumonic pasteurellosis. Vet Immunol Immunopath 1985; 1 : 265-78 . 13 . Slocombe RF, Ingersoll MR, Derksen FJ, Robinson NE . Importance of neutrophils in the pathogenesis of acute pneumonic pasteurellosis in calves . Am J Vet Res 1985; 46 : 2253-8. 14 . Braider MA, Walker RD, Hopkins FM, Schultz TW, Bowersock TL . Pulmonary lesions induced by Pasteurella haemolytica in neutrophil sufficient and neutrophil deficient calves . Can J Vet Res 1987 ; 52 : 205-9. 15 . Czuprynski CJ, Hamilton HL, Noel EJ . Ingestion and killing of Pasteurella haemolytica Al by bovine neutrophils in vitro . Vet Microbiol 1987 ; 14 : 61-74. 16 . Squire PG, Smiley DW, Coskell RG . Identification and extraction of Pasteurella haemolytica membrane proteins. Infect Immun 1984 ; 45 : 667-73 . 17 . Mikami T, Nagase T, Matsumoto T, Suzuki S, Suzuki M . Gelation of limulus amoebocyte lysate by simple polysaccharides . Microbial Immunol 1982; 26 : 403-9 . 18 . Confer AW, Simons KR . Effects of Pasteurella haemolytica lipopolysaccharide on selected functions of bovine leukocytes . Am J Vet Res 1986; 47 :154-7 . 19 . Greer CN, Shewen PE. Automated colorimetric assay for the detection of Pasteurella haemolytica leucotoxin . Vet Microbiol 1986; 12 : 33-42 . 20 . Noel EJ, Israel BA, Letchworth GJ, Czuprynski CJ . Incubation of bovine neutrophils with bovine herpesvirus-1 does not influence their interaction with Pasteurella haemolytica Al in vitro . Vet Immunol Immunopath 1988 ; 19 : 273-84 . 21 . Rest RF, Farrell CF, Naids FL. Mannose inhibits the human neutrophil oxidative burst . J Leuk Biol 1988 ; 43 : 158-64. 22 . Onderdonk AB, Kasper DC, Cisneros RL, Bartlett JG . The capsular polysaccharide of Bacteroides fragilis as a virulence factor : comparison of the pathogenic potential of encapsulated and unencapsulated strains . J Infect Dis 1977 ; 136 : 82-9 . 23 Connolly JC, McLean C, Tabaqchali S . The effect of capsular polysaccharide and lipopolysaccharide of Bacteroides fragilis on polymorph function and serum killing . J Med Microbiol 1984; 17 : 259-71 . 24 . Gonzales-Rayos C, Lo RYC, Shewen PE, Beveridge TJ . Cloning of a serotype-specific antigen from Pasteurella haemolytica Al . Infect Immun 1986 ; 53 : 505-10. 25 . Gentry MJ, Confer AW, Craven RC . Effect of repeated in vitro transfer of Pasteurella haemolytica Al on encapsulation, leukotoxin production and virulence . J Clin Microbiol 1987 ; 25 :142-5. 26 . Lessley BA, Confer AW, Mosier DA, et a! . Saline-extracted antigens of Pasteurella haemolytica : Separation by chromatofocusing, preliminary characterization and evaluation of immunogenicity . Vet Immunol Immunopath 1985; 10 : 279-96 . 27 . Durham JA, Confer AW, Mosier DA, Lessley BA . Comparison of the antigens associated with saline solution, potassium thiocyanate, and sodium salicylate extracts of Pasteurella haemolytica serotype 1 . Am J Vet Res 1986 ; 47 :1946-51 . 28 . Townsend EL, Maheswaran SK, Leiningr JR, Ames TR . Detection of immunoglobulin G to Pasteurella haemolytica capsular polysaccharide by enzyme-linked immunosorbent assay . J Clin Microbiol 1987 ; 25 : 242-7 . 29 . Whitley L, Leininger JR, Maheswaran SK, Ames TR . Abstract 153, 67th Annual Conference of Research Workers in Animal Disease, 1986 . Chicago IL. 30. Roth JA, Kaeberle ML . Evaluation of bovine polymorphonuclear leukocyte function . Vet Immunol Immunopath 1981 ; 2:157-74 . 31 . Haslett C, Guthrie LA, Kopaniak MM, Johnston RB Jr, Henson PM . Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide . Am J Pathol 1985; 119 : 101-10. 32 . Falk W, Goodwin RH Jr, Leonard EJ . A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration . J Immunol Methods 1980 ; 33 : 239-47 .