Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F

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1 Iowa State University From the SelectedWorks of Mark R. Ackermann December, 1995 Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F Richard B. Rimler, United States Department of Agriculture Karen B. Register, United States Department of Agriculture Tibor Magyar Mark R. Ackermann, United States Department of Agriculture Available at:

2 veterinary microbiology Veterinary Microbiology 47 ( 1995) Influence of chondroitinase on indirect hemagglutination titers and phagocytosis of Pasteurella multocida serogroups A, D and F Richard B. Rimler a-*, Karen B. Register a, Tibor Magyar b, Mark R. Ackermann a a U.S. Department of Agriculture, Agricultural Research Service, Avian Diseases Research Unit. National Animal Disease Cenfer, P.O. Box 70, Ames, Iowa, 50010, USA Veterinary Medical Research Institute, Hungarian Academy of Sciences, P.O. Box 18, H-1581 Budapest, Hungary Received 14 March 1995; accepted 23 May 1995 Abstract Capsules of Pasteurella multocida serogroups A, D and F contain mucopolysaccharides which block antigenic determinants and prevent phagocytosis. In this study, capsules of serogroup A, D and F strains of P. multocida were depolymerized by enzyme treatment. Capsule depolymerization of serogroup D and F strains with chondroitinase increased indirect hemagglutination (IHA) test titers and enhanced phagocytosis by swine neutrophils. Capsule depolymerization of serogroup A strains with hyaluronidase increased IHA titers, but depolymerization with chondroitinase did not. When serogroup A strains were treated with a combination of chondroitinase and hyaluronidase, IHA test titers were lower than titers of the same strains treated with hyaluronidase alone. Combined enzyme treatment of serogroup D strains resulted in II-IA test titers similar to those of chondroitinase treatment alone. Keywords: Pasteurella multocida; Serology: Phagocytosis; Capsule; Chondroitinase 1. Introduction Pasteurella multocida is a Gram-negative bacterial pathogen that produces septicemic and respiratory diseases in both domesticated and wild animals. Upon initial isolation, P. multocida organisms most often are capsulated. Serogroup specific antigens are associated * Corresponding author, USDA/ARS/MWA, National Animal Disease Center, Ames, Iowa 50010, USA, Tel.: , Fax: SSDIO (95)00127-l

3 288 R.B. Rider et al. / Veierinary Microbiology 47 (1995) with capsulated P. multocida, and 5 distinct serogroups (A, B, D, E, F) havebeenrecognized by indirect hemagglutination (IHA) tests (Rimler and Rhoades, 1989). Capsulated strains of P. multocida belonging to serogroups A, D and F are not readily phagocytized in nonimmunized animals. Laboratory passage of P. multocida can result in spontaneous dissociation, yielding non-capsulated variants which exhibit a loss or reduction in virulence. These non-capsulated variants do not react in the IHA test for serogroup specific antigen and are usually readily phagocytized. The inability of P. multocida serogroups A, D and F to be phagocytized by cells from non-immunized animals is associated with heat-stable, serologically-inert substances of the bacterial capsule. With P. multocida serogroup A, this substance is hyaluronic acid (Rimler and Rhoades, 1989). Capsule depolymerization of serogroup A strains with hyaluronidase results in an increase in susceptibility to phagocytosis (Anderson et al., 1984; Harmon et al., 1991; Maheswaran and Theis, 1979). Additionally, hyaluronidase treatment results in enhanced IHA test titers (Carter, 1972). The capsules of serogroups D and F are not depolymerized by hyaluronidase treatment, and the mechanisms which inhibit phagocytosis are unknown. A recent finding has shown that the capsules of serogroups A, D and F can be depolymerized by chondroitinase AC (Rimler, 1994). This finding indicated that the capsules of serogroups D and F contain mucopolysaccharides similar to hyaluronic acid found in the capsule of serogroup A strains. The purpose of this study was to determine whether chondroitinase AC depolymerization of the capsules of P. multocida results in a) increased IHA titers of serogroups A, D and F, and b) enhanced phagocytosis of serogroups D and F. 2. Materials and methods Bacteria Twenty-eight capsulated strains of P. multocida were used (Table 1). Capsule serogroup was determined by IHA tests (Rimler and Brogden, 1986) and somatic serotype was determined by gel diffusion precipitin tests (Heddleston et al., 1972). Capsulated and noncapsulated variants, obtained by spontaneous dissociation, of a toxigenic strain (P-4533; serotype D:3) and non-toxigenic strain (P-4218; serotype F:3) of P. multocida were used for phagocytosis studies. The capsules of all strains were depolymerized by chondroitinase AC, whereas only the capsules of serogroup A strains were depolymerized by hyaluronidase as determined by a disk diffusion method (Rimler 1994). Antisera Specific antisera against capsular serogroups A, D, and F for use in IHA tests were made in pasteurella-free rabbits, as described previously (Rimler and Brogden, 1986). The antisera were heat-inactivated and adsorbed with turkey red blood cells (TRBC) to remove heterophile antibodies. IHA Tests An 18 h culture was grown (37 C) on two dextrose starch agar plates (Baltimore Biological Laboratories, Rockville, MD) and suspended in 2.0 ml of PBS. The suspension was divided into four 0.5 ml aliquots. A 0.5 ml volume of phosphate-buffered saline (PBS),

4 R.B. Rimler et al. /Veterinary Microbiology 47 (1995) Table 1 Geometric mean indirect hemagglutination titers of 28 Pasreurella multocida strains with and without chondroitinase AC and hyaluronidase treatment Number of Strains Serotype Sources Enzyme Treatment None Chondroitinase Hyaluronidase Chondroitinase and Hyaluronidase 1 A:l 8 A:3 1 A:12 6 D:3 7 D:12 1 F:l 2 F:3 2 F:12 Chicken Bovine; Swine; Turkey Rabbit Rabbit; Swine Bovine; Rabbit; Swine Turkey Turkey; Sea Lion Turkey 32b ND ND ND ND ND ND *letter denotes capsule serogroup; number denotes somatic serotype bgeomettic mean titer expressed as reciprocal of serum dilution. PBS + Fluvobacterium heparinum chondroitinase AC (Sigma Chemical Co, St. Louis, MO; 0.5 U/ml), PBS + bovine testes hyaluronidase (Sigma Chemical Co.; 5 U/ml), or PBS + chondroitinase (0.5 U/ml) and hyaluronidase (5 U/ml) was added to an aliquot. The suspensions were incubated at 37 C for l-2 h. After heating to 100 C for 0.5 h, cells were removed by centrifugation. The supemate fluid was adjusted to 4.5 ml with PBS and a 0.5 ml volume of 10% washed TRBC was added. After incubation at 37 C for 1.5 h, sensitized-trbc were washed twice and adjusted to 0.5% (v/v) with PBS + 1.0% bovine serum albumin (PBSA). Serial 2-fold dilutions of antisera were made in PBSA, using a Microtiter system (Dynatech Laboratories Inc., Chantilly, VA). An equal volume of sensitized-trbc was added to each dilution, and Microtiter plates were incubated at room temperature for 1.5 h before readings were made. Phagocytosis (light microscopy) Capsulated, chondroitinase AC-treated, and non-capsulated variants of strains P-4533 and P-4218 were used for phagocytosis. Bacteria (grown 18 h on dextrose starch agar) were adjusted to 1 X lo9 organisms/ml of PBS. A 100 PL volume of bacteria were centrifuged and the pellets were resuspended in 50 PL of PBS. For chondroitinase AC treatment of capsulated bacteria, a 50 PL volume of PBS containing 0.5 U of enzyme was added. A 50 PL volume of PBS without enzyme was added to control samples of non-capsulated and capsulated bacteria. Following incubation at 37 C for 1 h, bacteria were washed twice and resuspended in 1.0 ml of PBS. Neutrophils (PMNs) harvested from whole normal swine blood (Roth and Kaeberle, 1981) were adjusted to 2 X lo7 cells/ml in PBS and PBS with 10% neonatal colostrum-deprived swine serum. The semm was negative for antibodies against P. multocida as determined by IHA and gel diffusion precipitin tests. A 100 PL amount of bacterial suspension was added to an equal volume of a PMN suspension and incubation was at 37 C for 1 h. PMNs were washed 3X and resuspended in 100 PL of buffy

5 290 R.B. Rider et al. / Veterinary Microbiology 47 (1995) coat-depleted normal swine blood. Thin film smears were made immediately on microscope slides and stained using a Diff-Quick stain set (Baxter Healthcare Corp., McGraw Park, IL). A total of 20 randomly selected PMNs were examined for ingestion of P. multocidu, and the numbers of bacteria per cell were counted. Two experiments were done for each strain. Flow cytometry Capsulated, chondoitinase AC-treated, and non-capsulated strains P-4533 and P-4218 were washed and treated as above. The bacteria were fluorescent labeled with carboxyfluorescein diacetate (Register et al., 1994) and added to swine PMNs in PBS and PBS with 10% neonatal colostrum-deprived swine serum at a ratio of 50: 1. PMN-associated fluorescence was determined immediately following the uptake period in a Coulter EPICS 752 flow cytometer. Approx. 15,000 events per sample were collected; samples were set up in duplicate or triplicate. 3. Results IHA Tests IHA test reactions between a rabbit antiserum and antigen sensitized-trbc were specific; reactions with more than one serogroup antiserum did not occur. Treatment with chondroitinase, hyahuonidase, or combinations of both enzymes did not affect test specificity. The data in Table 1 shows the influence of enzyme treatments on IHA titers. Chondroitinase treatment resulted in increased titers with serogroup D and F strains, but not serogroup A strains. Treatment with hyaluronidase resulted in increased IHA titers with serogroup A strains, but not with serogroup D strains. Treatment with combinations of both chondroitinase and hyaluronidase resulted in markedly diminished titers with serotype A strains as compared to hyaluronidase treatment alone, but serogroup D strains were relatively unaffected. Phagocytosis of P. multocida by PMNs Fig. la,b,c show PMNs and phagocytosis of capsulated, enzyme-decapsulated, and noncapsulated P. multocida, respectively. The data showing phagocytosis of toxigenic strain P-4533 and non-toxigenic strain P-4218 as determined by light microscopy is shown in Table 2. Capsulated bacteria of either strain were poorly phagocytized in the presence or absence of serum. In contrast, non-capsulated bacteria of either strain were readily phagocytized. After capsule depolymerization by chondroitinase, phagocytosis of both strains increased. The presence of native or heat-inactivated serum resulted in marked increased phagocytosis of enzyme-treated P-4533, but not enzyme-treated P Heat inactivation of serum and whether a strain was toxigemc did not affect phagocytosis. Except that heatinactivated serum was not tested, data from the flow cytometry experiments (Table 3) supported findings from light microscopy.

6 R.B. Rider et al. /Veterinary Microbiology 47 (1995) Fig. 1. Ph Strai m P-4 mis of capsulated (a), chondroitinase-treated (b), and non-capsulated (c swine PMNs. Pasteurella 1 multoc kil

7 292 R.B. Rimler et at. / Veterinary Microbiology 47 (1995) Table 2 Phagocytosis of capsulated (C), non-capsulated variant (NC), and chondroitinase AC-treated (CT) P. multocida strains P-4533 (serotype D:3) and P-4218 (serotype F:3) by swine PMNs as determined by light microscopy No Serum Avg. # bacteria per PMN % PMN Phagocytic Serum Heat-inactivated serum Avg. # % PMN Avg. # % PMN bacteria per Phagocytic bacteria per Phagocytic PMN PMN Strain P-4533 c 0.75 NC 12.1 CT 2.2 Strain P-42 1 Sb C 0.6 NC 8.63 CT JO Toxigenic strain bnon-toxigenic strain Table 3 Phagocytosis of capsulated (c), non-capsulated variant (NC), and chondroitinase AC-treated (CT) P. multocida strain P-4533 (serotype D:3) and P-4218 (serotype F:3) by swine PMNs as determined by cell-associated fluorescence. Mean cell-associated fluorescence of C in each experiment was set to a baseline of 100% and individual samples were. compared with these values Mean percent cell-associated fluorescence No serum With serum Strain P-4533 C NC CT Strain P ( < O.OOO1)d 493 ( 0.001) 177b(0.032) 306 (0.015) C 100 NC 147 (0.084) CT 108 ( 0.034) (0.05) 124 (0.023) Mean of 2 experiments, n = 5 bmean of 3 experiments, n = 8 Mean of 2 experiments, n = 6 dnumbers in parentheses are P values. 4. Discussion In a previous study ( Rimler, 1994)) several mucopolysaccharidases were tested for ability to depolymerize the capsules of serogroup A, D and F P. multocida. One enzyme, chondroitinase AC, was active against capsules of all serogroups. This finding suggested that chondroitinase AC might be useful for serotyping by improving the sensitivity of the IHA

8 R.B. Rimler et al. /Veterinary Microbiology 47 (1995) test. The present study showed that treatment of serogroup D and F strains with this enzyme resulted in enhanced sensitivity of the IHA test without loss of specificity. However, no improved sensitivity resulted after treatment of serogroup A strains, even though the enzyme was active in depolymerizing their capsules. In contrast, hyaluronidase enhanced the sensitivity of the IHA test with serogroup A strains as shown here and previously described by Carter ( 1972). Surprisingly, treatment of serogroup A strains with both chondroitinase AC and hyaluronidase resulted in diminished IHA test sensitivity when compared to hyaluronidase treatment alone. The reason for diminished sensitivity was not determined, but the evidence suggests that chondroitinase AC might destroy certain antigenic determinants associated with the specific capsule antigen of these strains. Previous reports have shown that phagocytosis of serogroup A strains of P. multocida is enhanced after capsule depolymerization by hyaluronidase (Anderson et al., 1984; Harmon, et al., 1991; Maheswaran and Theis, 1979). The present study showed that non-capsulated variants, derived from a toxigenic serogroup D strain and a non-toxigenic serogroup F strain were readily phagocytized by normal swine PMNs. In contrast, the parent capsulated strains were not readily phagocytized. Capsule depolymerization of these parent strains with chondroitinase AC increased phagocytosis. However, for the serogroup D strain, enhanced phagocytosis was dependent, in part, on the presence of serum. The toxin producing ability of the serogroup D strain did not appear to intluence phagocytosis. The data in this and previous studies (Anderson et al., 1984; Harmon et al., 1991; Maheswaran and Theis, 1979)) indicate that a mucopolysaccharide capsule is important in preventing phagocytosis. However, other factors, such as anti-phagocytic proteins, (Ryu et al., 1984; Truscott and Hirsh, 1988), may contribute to this effect. Acknowledgements The authors greatly appreciate the excellent technical assistance of Ms. Rim Driftmier. References Anderson, L.C., Rush, H.G. and Gloriso, J.C., Strain differences in the susceptibility and resistance of Pasteurella multocida to phagocytosis and killing by rabbit polymorphonuclear neutrophils. Am. J. Vet. Res., 45: 1193-l 198. Carter, G.R., Improved hemagglutination test for identifying type A strains of Pasteurella multocida. Appl. Microbial., 24: Harmon, B.G., Glisson, J.R., Latimer, K.S., Steffens, W.L. and Nmmally, J.C., Resistance of Pasteurella multocida A:3,4 to phagocytosis by turkey macrophages and heterophils. Am. J. Vet. Res., 52: Heddleston, K.L., Gallagher, J.E. and Rebers, P.A., Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis., 16: Maheswaran, SK. and Theis, E.S., Influence of encapsulation on phagocytosis of Pasteurella multocida by bovine neutrophils. Infect. Immun., 26: Register, K.B., Ackermann, M.R. and Kehrli, Jr., M.E., Non-opsonic attachment of Bordetella bronchiseptica mediated by CD1 l/cd18 and cell surface carbohydrates. Microbial. Pathogen., 17: Rimler, R.B., Presumptive identification of Pasteurella multocida serogroups A, D and F by capsule depolymerization with mucopolysaccharidases. Vet. Rec., 134:

9 294 R.B. Rimier et al. /Veterinary Microbiology 47 (1995) Rimler, R.B. and Brogden, K.A., Pasteurella multocido isolated from rabbits and swine: serologic types and toxin production. Am..I. Vet. Res., 47: Rimler, R.B. and Rhoades, K.R., Pasteurella multocida. In: C. Adlam and J.M.Rutter, eds.), Pasteurella and Pasteurellosis, Academic Press, London, pp Roth, J.A. and Kaeberle, M.L., Evaluation of bovine polymorphonuclear leukocyte function. Vet. Immunol. Immunopathol., 2: Ryu, H., Kaeberle, M.L., Roth, J.A. and Griffetb, R.W., Effect of type A Pasteurella multocida fractions on bovine polymorphonuclear leukocyte functions. Infect. Immun., 43: Truscott, W.A. and Hirsh, D.C., Demonstration of an outer membrane protein with antiphagccytic activity from Pasteurella multocida of avian origin. Infect. Immun., 56: