Veterinary Microbiology, 25 ( 1990 ) 241-251 Elsevier Science Publishers B.V., Amsterdam
Resistance to host immune defense mechanisms afforded by capsular material of Pasteurella haemolytica, serotype 1 C.H. Chae a, M.J. Gentry a'*, A.W. C o n f e r b a n d G.A. A n d e r s o n a'** aDepartment of Veterinaey Science, University of Nebraska, Lincoln, NE 68583-0905, U.S.A. bDepartment of Veterinary Pathology, Oklahoma State University, Stillwater, OK 74078, U.S.A. (Accepted 12 March 1990)
ABSTRACT Chae, C.H., Gentry, M.J., Confer, A.W. and Anderson, G.A., 1990. Resistance to host immune defense mechanisms afforded by capsular material ofPasteurella haemolytica, serotype 1. Vet. Microbiol., 25: 241-251. Selected serum-mediated host immune defense mechanisms against Pasteurella haemolytica were studied using encapsulated and decapsulated organisms. When the capsular material was removed from P. haemolytica, it became more susceptible to serum agglutination, complement-mediated serum killing, and phagocytosis by polymorphonuclearleukocytes. When encapsulated organisms were used, phagocytosis was enhanced by antibodies to capsular material produced by vaccination of calves with any of three P. haemolytica vaccines. The serum bactericidal activity, however, was not facilitated by increased levels of anticapsular antibody in vaccinated cattle. By contrast, when decapsulated organisms were used, vaccination enhanced both the bactericidal and opsonizing capacities of sera from the calves. These studies indicate that capsular material should be considered a principal virulence factor for P. haemolytica.
Bovine pneumonic pasteurellosis, also called shipping fever, is a severe fibrinous pleuropneumonia of feedlot cattle (Yates, 1982). Pasteurella haemolytica, serotype 1 is the major causative organism, along with other factors such as stress and bovine respiratory viruses (Yates 1982; Confer et al., 1988 ). The complicated and multifactorial etiology of the disease makes it difficult *To whom reprint requests should be addressed, present address: Creighton University, Section of Infectious Diseases, Veterans Administration Medical Center, 4101 Woolworth Ave., Omaha, NE 68105, U.S.A. **Present address: CEVA Laboratories, INC., 12300 Santa Fe Drive, Lenexa, KS 66215, U.S.A. ~Journal Series No. 9009 from the Agricultural Research Division, University of Nebraska.
© 1990 - - Elsevier Science Publishers B.V.
C.H. CHAE ET AL.
to determine the specific virulence mechanisms of P. haemolytica or to understand what constitutes effective immunity to the disease. P. haemolytica is encapsulated during the logarithmic phase of growth in vitro, but the capsule is lost as the culture ages (Corstvet et al., 1982). The loosely associated capsular material can be removed from logarithmic-phase cultures by gentle heat treatment without substantial loss of viability (Gentry et al., 1982). P. haemolytica also is encapsulated within the lung tissue of infected cattle (Morck et al., 1988 ). Colonization of pulmonary alveolar spaces with P. haemolytica through attachment to type 1 pneumocytes is an important initial step in the pathogenesis ofpneumonic pasteurellosis, and attachment may be facilitated by P. haemolytica's capsular glycocalyx and/or fimbriae (Morck et al., 1988 ). Capsules and fimbriae may protect a number of bacteria and fungi from immune host defense mechanisms, including phagocytosis and complement-mediated bactericidal activity (Costerton et al., 1981 ). Encapsulated logarithmic-phase P. haemolytica serotype 1 cells resisted phagocytosis by bovine alveolar macrophages (BAM), whereas stationaryphase, noncapsulated organisms were phagocytized by BAM, particularly in the presence of specific opsonins (Walker et al., 1984). Although BAM are the first line of defense against pneumonic pasteurellosis (Markham and Wilkie, 1980), polymorphonuclear leukocytes (PMN) may be the most important phagocytes within the P. haemolytica-infected lung, because after experimental challenge with the organism, PMN rapidly outnumbered the BAM (Walker et al., 1985 ). Czuprynski et al. (1987) reported that in the presence of specific opsonins, PMN ingested and killed both logarithmic-phase and stationary-phase cultures of one P. haemolytica serotype 1 strain. In the present study, the effect of capsular material in protecting P. haemolytica from selected host immune defense mechanisms was examined. Serum agglutination, complement-mediated serum killing, and phagocytosis by PMN were measured for encapsulated and decapsulated organisms using sera from five unvaccinated controls and 15 calves that had each been immunized with one of three different P. haemolytica vaccines. Decapsulated logarithmic-phase bacteria were used rather than stationary-phase, noncapsulated ones to minimize other variables between the organisms. MATERIALS AND METHODS
Microorganisms The P. haemolytica serotype 1 used was isolated originally from a feedlot calf. For each experiment, several isolated colonies were inoculated into brain heart infusion broth (BHI; Difco, Detroit, MI). After 5 h of incubation at 37 °C in a shaker bath, the logarithmic-phase culture was washed and resuspended in phosphate buffered saline (PBS) to an optical density (OD65o) of
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Fig. 1. Photomicrograph of Maneval-stained organisms from a 6-h culture of P. haemolytica ( × 900). (A) Encapsulated bacteria, before extraction procedure; (B) same culture decapsulated by heating in PBS for 1 h at 41 °C.
0.12 (approximately 1 × 108 colony-forming units ( C F U ) / m l ) . To prepare decapsulated organisms, a portion of the 5-h PBS suspension was incubated in a shaker bath at 41 ° C for 1 h with gentle aggitation as described previously (Gentry et al., 1982). The organisms were washed once and then resuspended in PBS to an OD65o equal to that of the encapsulated culture. The actual number of C F U / m l in each suspension was determined by a spot plate counting method. Decapsulation was confirmed in each experiment by a Maneval Stain (Corstvet et al., 1982), as shown in Fig. 1. Sefa
Twenty sera were collected from beef calves used in vaccine trials that were conducted at Oklahoma State University and reported previously (Confer et al., 1985a, 1987). Four groups of five sera each were taken from calves vaccinated with: ( 1 ) PBS; (2) 5 ml of live P. haemolytica ( 1 × l 0 9 C F U / m l ) , (3) 5 ml of a formalin-killed P. haemolytica bacterin (equivalent to 1 x 109 C F U / m l ) in an aluminum hydroxide adjuvant; (4) 5 ml of the same bacterin mixed with Freund's incomplete adjuvant (FIA). Calves were vaccinated twice (on days 0 and 7), and then challenged on day 21 by a transthoracic intrapulmonic inoculation of live P. haemolytica organisms (Panciera and Corstvet, 1984). Sera used in the present study were collected on the day of challenge. Serum antibody levels to a protein-carbohydrate subunit ofP. haemolytica surface material were determined previously by an enzyme-linked immunosorbent assay and reported elsewhere (Confer et al., 1985a,b). The group mean values _+standard deviations for anticapsular antibody of the sera
C.H. CHAE ET AL.
used in the present study were as follows: PBS control group, 0.204 _+0.126; bacterin with AI(OH)3 adjuvant group, 0.523_+0.262; bacterin with FIA group, 0.845 _+0.342; live organism group, 0.843 _+0.403. Values were significantly higher ( P < 0.05 ) for the last two vaccinated groups than for the PBS control group. There were no significant differences ( P > 0.05 ) in antibody values among the three groups of vaccinated animals.
Direct bacterial agglutination (DBA) test Two-fold serial dilutions of serum were made in PBS in 12 × 75 m m tubes. An equal volume of a formalin-killed suspension of either encapsulated or decapsulated P. haemolytica organisms (OD525 = 1.0) was added to each tube. The tubes were incubated at 37 °C for 24 h and were then examined for macroscopic agglutination using a dissecting microscope. The agglutination titer for each serum was determined as the reciprocal of the highest dilution that exhibited agglutination. Serum bactericidal assay Complement-mediated serum killing was determined by measuring the change in the n u m b e r of C F U / m l in a P. haemolytica culture incubated with serum and a complement source. All sera were heat-inactivated at 56 °C for 30 min. Test mixtures were prepared by adding 0.4 ml of a suspension of either encapsulated or decapsulated organisms to a tube containing 0.05 ml of test serum, 0.05 ml of PBS, and 0.1 ml of serum from a colostrum-deprived calf (containing four hemolytic units of complement ). Immediately after vigorous mixing (0 time sample), and after 30 min of incubation at 37°C in a water bath, 0.1 ml aliquots of the suspensions were diluted into 9.9 ml of cold (4 ° C ) PBS. All tubes were maintained in an ice bath until they could be further diluted and plated for counting by a spot plate method. The percentage of killing was calculated by the following formula: % killing= ( ( C F U / m l at 30 m i n - C F U / m l at time 0 ) / C F U / m l at time 0) × 100. Phagocytosis assay Bovine P M N were isolated from sodium citrate-treated peripheral blood from a donor cow. After centrifugation at 1000 × g for 40 min, the plasma and buffy coat were removed and discarded. An equal volume of Hank's balanced salt solution without Ca 2÷ and Mg 2÷ (HBSS; GIBCO, Grand Island, NY) was added to the remaining cell pellet, and the centrifugation and buffy coat removal steps were repeated. The remaining leukocytes were separated from the erythrocyte pellet by two hypotonic lysis treatments (Gentry et al., 1985 ). The final leukocyte pellet was resuspended in HBSS containing Ca 2÷ and Mg 2÷ (complete HBSS) to a concentration of 1 × 107 cells/ml. Differential
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counts on the suspensions typically revealed 93 to 95% PMN, 3 to 4% eosinophils, and 2 to 3% lymphocytes, with > 99% viability as determined by trypan blue exclusion. To prepare radioactively labeled bacteria, the organisms were grown to logarithmic phase in BHI broth containing 5 ~tCi/ml of [3H ]-thymidine (Amersham, Arlington Hts, IL). Organisms were collected by centrifugation, washed twice in PBS, and suspended in PBS. A portion of the suspension was then decapsulated as described above, and both the encapsulated and decapsulated organisms were resuspended in complete HBSS to an OD650 of 0.12. The phagocytosis test mixture consisted of 25/tl of serum, 50/~1 of bacterial suspension, 75/zl of complete HBSS, and 100/tl of PMN suspension added to triplicate wells of 96-well tissue culture plates. Control wells for each serum, also prepared in triplicate, did not contain phagocytes. The total bacteriaassociated counts per minute (CPM) added to the wells was determined by spotting 50/~1 of labeled bacterial suspension directly onto paper disks for counting. The plates were incubated at 37 °C for 30 min and then centrifuged at 150Xg for 5 min before the supernatants were removed from each well. The remaining cell pellets were washed twice and suspended in complete HBSS. Well contents were harvested onto filter paper disks using an automated cell harvester (Skatron, Rockville, MD) and were counted in a scintillation counter (Packard, Downers Grove, IL). The percentage ofphagocytosis was calculated by the following formula: % phagocytosis= ( ( C P M test wells-CPM control wells)/(total bacterial CPM a d d e d - C P M control wells) ) × 100.
Statistical analys& Differences between encapsulated and decapsulated organisms tested in each of the three assays were compared by paired Student t tests. Differences among the vaccine groups for antibodies to capsular material and values for the bactericidal and phagocytosis assays were determined by Duncan's multiple contrast tests. RESULTS
Direct bacterial agglutination test For sera from calves in the control group, there was no significant difference between the mean DBA titers for encapsulated and decapsulated organisms ( P > 0.05, Table 1 ). For sera from vaccinated calves pooled from all three vaccine groups, however, the DBA titer for decapsulated organisms was significantly higher ( P < 0.05 ) than that for encapsulated organisms. For both encapsulated and decapsulated organisms, vaccination of the animals significantly enhanced ( P < 0.05 ) their serum agglutination titers.
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TABLE 1 Direct bacterial agglutination assay Treatment group
No. of sera
PBS controls Vaccinatedb
Mean DBA titer _+SD" Encapsulated organisms
26.7 +_9.2 58.0 _+34. I c
24.0 + 13.9 98.0+_ 74.5 ''d
"Direct bacterial agglutination titers determined as reciprocal of the highest 2-fold serum dilution effecting macroscopic agglutination. bAnimals were vaccinated either with live bacteria or formalin-killed organisms combined with AI (OH)3 or Freund's incomplete adjuvant. CResult is significantly greater (P< 0.05 ) for sera from vaccinated animals than for sera from control animals. dResult is significantly greater (P< 0.05 ) for decapsulated organisms than for encapsulated ones when using sera from vaccinated animals.
BACTERIN B b
LIVE VACCINE c
~ ~oo ~ ~
8o 40 2O
Fig. 2. Change in viability (% change C F U / m l ) of suspensions of decapsulated  or encapsulated • organisms after 30 min of incubation in PBS containing 10% serum from cattle vaccinated with PBS or with (a) bacterin with AI(OH)3 adjuvant; (b) bacterin with Freund's incomplete adjuvant; or (c) 5 × 109 CFU of live P. haemolytica. Bars indicate group mean values with standard deviations.
S e r u m bactericidal a s s a y Encapsulated organisms were completely resistant to killing by each serum t e s t e d f r o m t h e c o n t r o l a n d v a c c i n a t e d g r o u p s , ( F i g . 2 ). T h e n u m b e r o f C F U / m l i n e n c a p s u l a t e d c u l t u r e s i n c r e a s e d d u r i n g t h e 30 m i n i n c u b a t i o n p e r i o d f o r all s a m p l e s . D e c a p s u l a t e d o r g a n i s m s , h o w e v e r , w e r e k i l l e d b y a l l s e r a f r o m vaccinated, but not control, calves. For decapsulated organisms, there were no significant differences in killing capacity of sera from the three vaccinated groups (P> 0.05).
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TABLE2 Phagocytosis assay of encapsulated and decapsulated P. haemolytica by bovine polymorphonuclear leukocytes after treatment with sera from control or vaccinated calves Vaccine group
PBScontrols Bacterin A c Bacterin B d Live vaccine e
No. of sera
5 5 5 5
Mean % phagocytosis _+SD a'b Encapsulated organisms
8.1 +4.6 f 20.0_+4.2 19.2 + 7.0 19.6-+ 5.8
36.9_+ 8.9 44.3_+ 6.9 54.1 + 13.4g 52.1 _+ 7.4 g
aPercentage phagocytosis determined as percentage of total radioactive bacteria which became phagocyte-associated after 30 min of incubation. bResults are significantly greater ( P < 0.05) for decapsulated than for encapsulated organisms within each treatment group. CBacterin with aluminum hydroxide adjuvant. dBacterin with Freund's incomplete adjuvant. eAnimals were vaccinated intramuscularly with 5 ml of 1 × 10 9 C F U / m l of live P. haemolytica organisms. fResults are significantly less ( P < 0.05 ) for the control group than for each of the vaccinated groups when using encapsulated organisms. gResults are significantly greater ( P < 0 . 0 5 ) than for the control group when using decapsulated organisms.
Phagocytosis assay Using sera from each of the four treatment groups, decapsulated organisms were phagocytized significantly better ( P < 0 . 0 5 ) than encapsulated organisms (Table 2 ). For encapsulated organisms, sera from each group of vaccinated calves opsonized the bacteria significantly better ( P < 0.05 ) than did sera from control calves. There were no significant differences ( P < 0.05 ) in opsonizing capacity of sera from the vaccinated groups using encapsulated organisms. When decapsulated organisms were used, sera from calves vaccinated with live organisms or the bacterin in FIA opsonized the organisms significantly better ( P < 0.05 ) than did sera from control calves. DISCUSSION
The capsular polysaccharides of certain bacteria may be important in resistance to host immune defense mechanisms (Jann and Westphal, 1975; Woolcock, 1985 ). Encapsulation therefore has been related to virulence for a number of Gram positive and Gram negative bacteria (MacLeod and Krauss, 1950; Melly et al., 1974; Maheswaran and Thies, 1979; Sawata and Kume, 1983). In the present study, encapsulated organisms were more resistant than decapsulated ones to agglutination by sera from vaccinated but not control
C.H. C H A E ET AL.
calves. It has been suggested that the presence of capsular material interferes with bacterial agglutination by inhibiting lattice formation between adjacent organisms (Ceppellini and Landy, 1963). Removal of the capsular material would allow agglutination of the bacteria by antibodies to somatic antigens present in sera from vaccinates. Encapsulated P. haemolytica also were more resistant than decapsulated ones to complement-mediated serum killing. One c o m m o n explanation for this is that capsular material may form a barrier between the membrane attack complex (complement components C5 through C9) and the bacterial surface, thus preventing an effective lytic lesion even when anticapsular antibodies are present (Howard and Glynn, 1971; Wright and Levine, 1981 ). In the present study, encapsulated P. haemolytica were completely resistant to the bactericidal action of bovine serum and complement even though many of the sera had high antibody titers to a capsular component of the organism. MacDonald et al. ( 1983 ) reported serum killing of logarithmic-phase P. haemolytica serotype 1 by normal adult bovine sera. In those experiments, 50% unheated fresh serum and approximately 6.7 X 103 bacteria/ml, were used. Dilution of the sera or heat inactivation resulted in a loss of bactericidal activity. In the present study, 10% serum and approximately 1 X 108 bacteria/ ml were used. These variations in methods may explain the apparent difference in complement-mediated serum killing of P. haemolytica shown in the two studies. Decapsulated P. haemolytica were susceptible to complementmediated killing by sera from vaccinated but not control calves. Increased susceptibility of bacteria to serum-mediated bactericidal activity was shown for capsule-deficient mutants of other Gram negative bacteria (Branefors and Dahlberg, 1980; Sandstrom et al., 1988 ). Capsular material may inhibit phagocytosis by PMN, and opsonization of certain encapsulated organisms was shown to require specific anticapsular antibody (Van Dijk et al., 1977; Karakawa et al., 1988 ). In the present study, encapsulated P. haemolytica were relatively resistant to phagocytosis by PMN. However, phagocytosis was enhanced by sera from vaccinated calves. When the capsular material was removed from P. haemolytica, the organisms became more susceptible to phagocytosis by P M N after opsonization by sera from all four groups of animals. Increased phagocytosis has been shown for other decapsulated or noncapsulated m u t a n t bacteria when compared to encapsulated strains (Maheswaran and Thies, 1979; Wilkinson et al., 1979; Eftekhar and Speert, 1988; Sandstrom et al., 1988). In the present study, it is not clear why considerable phagocytosis of decapsulated organisms occurred when using sera from the PBS control group. It may be that sera from those calves contained cross-reacting antibodies to somatic antigens of other Gram negative bacteria which were able to opsonize the decapsulated P. haemolytica. The data in the present study indicate that phagocytosis of encapsulated P. haemolytica by PMN was increased, whereas complement-mediated serum
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bactericidal activity against encapsulated organisms was not affected by the presence o f serum containing antibody to capsular material. This m a y be because 10 times more IgM or IgG molecules can be required to kill certain organisms than are needed to opsonize t h e m ( H o w a r d and Glynn, 1971 ). In addition, bactericidal activity requires all nine complement components (C 1 through C9), whereas phagocytosis requires only C1, 4, 2, and 3 (Johnson et al., 1969; H o w a r d and Glynn, 1971 ). The differences seen between the two defense mechanisms could be that the capsule might interfere with the functioning o f the later more than the earlier components of the complement reaction. In conclusion, the capsule o f P . haemolytica was important in its resistance to selected host i m m u n e defense mechanisms and should, therefore, be considered a principal virulence factor for this organism. Sera from vaccinated calves that contained specific antibody to capsular material facilitated PMNm e d i a t e d phagocytosis of the bacterium. These results support the recent report that serum anticapsular antibodies m a y be o f particular importance in resistance to p n e u m o n i c pasteurellosis (Confer et al., 1989).
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