JOURNAL OF CLINICAL MICROBIOLOGY, JUlY 1991, P. 1328-1332 0095-1137/91/071328-05$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 29, No. 7
Detection and Enumeration of Toxin-Producing Pasteurella multocida with a Colony-Blot Assay TIBOR MAGYAR' AND RICHARD B. RIMLER2* Veterinary Medical Research Institute, Hungarian Academy of Sciences, H-1581 Budapest, Hungary,' and Avian Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, Iowa 500102 Received 31 December 1990/Accepted 2 April 1991
Colonies of toxin-producing Pasteurella multocida were detected with peroxidase-labeled monoclonal antibodies by a membrane assay. Examination of the specificity of the assay with 29 P. multocida cultures representing various geographic origins, hosts, and serotypes indicated that the test was specific for toxinproducing strains. No cross-reactions were observed with Bordetella species that can be associated with P. multocida in producing diseases in animals. A single membrane could be used to assay several isolated strains for toxin production or to enumerate toxin-producing colonies in mixed cultures. Toxin-producing P. multocida colonies were detected in primary cultures; hence, the assay appears to have good potential for widespread application with clinical samples. In the present report we describe an inexpensive and simple membrane assay with peroxidase-labeled MAb to recognize and enumerate toxin-producing P. multocida.
Progressive atrophic rhinitis of swine is a complex disease in which toxin-producing serogroup D Pasteurella multocida together with toxin-producing Bordetella bronchiseptica are recognized as the major etiologic agents (2, 18, 26). Although the dynamics of the interaction between these two agents in producing the disease are not yet understood, inoculation of pigs with cell extracts of toxin-producing P. multocida alone, either intranasally (11) or intraperitoneally (27), has reproduced the characteristic lesions of severe turbinate atrophy. These findings indicated that a toxin plays a central role in the disease. This role was confirmed when the characteristic lesions developed after intranasal (7) or intraperitoneal (4) inoculation of gnotobiotic pigs with protein toxin purified from serogroup D P. multocida. Although most reports describe toxin-producing serogroup D P. multocida, toxin-producing serogroup A organisms have also been isolated from swine (5, 8, 19), and the toxin from these organisms is either antigenically similar or identical to that from serogroup D organisms (9, 22). Besides affecting swine, recent research indicates that toxin-producing P. multocida organisms are involved in the etiology of naturally occurring progressive atrophic rhinitis of goats (1). Toxin-producing P. multocida has been isolated from rabbits, cattle, cats, dogs, and turkeys (6, 12, 17, 20, 22). However, the role of P. multocida toxin in species other than swine and goats has not been clarified. P. multocida toxin can be detected in cell extracts and culture filtrates by its lethality and lienotoxicity for mice (13, 15, 24) and production of dermonecrosis in guinea pig skin (5). Recognition of toxin-producing organisms by these methods is time-consuming, and the number of samples that can be processed is limited. In vitro techniques such as cytopathogenic effects in tissue culture (25), the agar overlay method (3), and an enzyme-linked immunosorbent assay with toxin-specific monoclonal antibodies (MAbs) (9) can recognize toxin-producing P. multocida. While these in vitro techniques allow testing of a larger number of suspect toxin-producing P. multocida isolates, they are cumbersome and require expensive equipment. *
MATERIALS AND METHODS
Bacteria. A total of 29 P. multocida strains representing various hosts, geographic origins, and serotypes were examined (Table 1). Typing of capsule serogroup antigen was done by passive hemagglutination tests as described previously (22). Somatic serotypes of these strains were determined by the gel diffusion precipitin test described by Heddleston et al. (10). B. bronchiseptica P-4609 and P-4607, a toxin-producing and a non-toxin-producing strain, respectively, from pigs affected by atrophic rhinitis (in Iowa), and Bordetella avium P-4148, a toxin-producing isolate from a turkey in Ohio, were used as control cultures. For examination of the potential of nonimmunological immunoglobulin G (IgG)-binding bacteria in producing falsepositive reactions, the Cowan 1 strain of Staphylococcus aureus (ATCC 12598) was included in the study. Preparation of samples. Bacterial strains were cultured on glucose starch agar plates and 5% bovine blood agar plates at 37°C. Colonies were examined in the membrane assay after 12, 16 to 18, and 48 h of growth. For enumeration experiments, growth was suspended in tryptose broth, and the suspension was adjusted to a density equal to a McFarland no. 1 nephelometer standard. Serial dilutions of the suspensions were made in tryptose broth and plated onto blood agar to result in an optimum of approximately 80 to 100 colonies per plate. Strains were tested in pure culture, except when mixed cultures of toxin-producing and non-toxin-producing P. multocida or B. bronchiseptica were prepared to examine the specificity of the assay. For examination of several individual strains, 10 or more cultures were streaked as separate 2-cm lines on the same agar plate. Antibodies. Mouse ascitic fluid containing IgG MAbs specific to the toxin of serogroup D P. multocida was made with hybridoma ot-DNT-1B2A3 and provided by lone Stoll, National Veterinary Services Laboratories, Science and
Corresponding author. 1328
TOXIN-PRODUCING P. MULTOCIDA
VOL. 29, 1991
TABLE 1. Lethal toxicities of filtered sonic extracts and colony reactions in a membrane assay of selected P. multocida isolates representing different hosts, origins, and serotypes Strain
P-4066 P-4067 P-3766 P-4671 P-4672 P-4673 P-5049 P-5050 P-5062 P-5066 P-5069 P-5070
Host
Geographic ongin
Rabbit Belgium Rabbit Belgium Rabbit Belgium Rabbit Michigan Rabbit Michigan Rabbit Michigan Swine Norway Swine Norway Swine Norway Swine Norway Swine Norway Swine Norway P-%1 Swine Maryland P-3018 Swine Brazil P-4531 Swine Sweden P-45331 Swine Sweden P-4533B Swine Sweden P-4976 Swine Norway P-4275 Swine Arkansas P-4980 Goat Norway P-5041 Goat Norway P-5042 Goat Norway P-5043 Goat Norway P-5044 Goat Norway P-5045 Goat Norway P-5046 Goat Norway P-5048 Goat Norway P-4123 Cattle Mali X-73 Chicken Maryland a MA, membrane assay.
Serotype
D:3, 4" D:3, 12 D: 12 D:3 D:3 D: 12 A:3 A:3 A:3, 4 A:3 A:3 A:3 D:3 D: 12 D:12 D:3 -:3c A:3 -:12
D:3 D:3, 4 D:3, D:3, D:3, D:3, D:4, A:3 E:5 A: 1
4, 12 4, 12 4 4 7, 12
Lethality for Reaction in mice MA
+ +
+ +
-
-
-
-
-
-
-
-
-
-
-
-
+ +
+ +
+
+
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
-
-
b The letter indicates capsule serogroup; the number(s) indicates reaction with serotype antiserum. c -, absence of a letter for the capsule group denotes a noncapsulated strain.
Technology, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa. Hybridoma a-DNT-1B2A3 (to be deposited with the American Type Culture Collection) was prepared with mice immunized with a toxoid. The toxoid, a gift from J. C. Frantz, Norden Laboratories, Inc., Lincoln, Nebr., was made by a proprietary procedure with toxin purified from the NOR-1 strain of P. multocida, as described by Martineau-Doize et al. (14). To purify MAb, the ascitic fluid was mixed 1.5:1 with Seroclear (Calbiochem, La Jolla, Calif.) and centrifuged. The supernatant was removed by aspiration, diluted with 4 volumes of Tris-NaCl buffer (0.05 M Tris, 0.15 M NaCI [pH 7.3]), and applied to a column (1.6 by 14 cm) of Avid Al gel (BioProbe, Tustin, Calif.) equilibrated with the same buffer. IgG MAb was eluted from the column with 3 M KSCN in Tris-NaCl buffer, dialyzed against Tris-NaCl buffer, and concentrated over an XM-50 membrane (Amicon Corp., Danvers, Mass.). The purified MAbs were labeled with horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) by the method of Nakane and Kawaoi (16). A block titration in a dot blot assay was done with purified P. multocida toxin (22) versus peroxidase-labeled MAb. The detection limit of a 1:250 working dilution of labeled MAb was 0.042 ag of purified toxin. Chicken antiserum specific for the lipopolysaccharide of P. multocida serotype 1 was prepared as described previously (21). Peroxidase-labeled goat anti-chicken IgG was
1329
from Kirkegaard and Perry Laboratories (Gaithersburg, Md.). Membrane assay. Modified nylon 66 (Nytran; Schleicher & Schuell, Keane, N.H.) or nitrocellulose (BA 85; Schleicher & Schuell) membrane circles were used for the assay. For colony transfers, the dry membranes were gently placed onto an agar surface, pressed lightly and evenly with an L-shaped glass rod, removed with forceps, and placed (colony side up) onto blotting paper saturated with 0.5 M NaOH. The colonies were allowed to lyse for about 5 min. After lysis, the membranes were transferred to blotting paper saturated with phosphate-buffered saline solution (pH 7.0) for neutralization. All steps thereafter were done in petri plates at room temperature with constant horizontal rotation at about 80 rpm. Nonreacted binding sites on the membranes were blocked by incubating them for 1 h in a solution (BS) that contained 1% casein (Fisher Chemical Co., Fair Lawn, N.J.), 0.05% Tween 20, 0.025 M Tris, and 0.192 M glycine in distilled water (pH 8.3). After blocking, membranes were incubated for 1 h with peroxidase-labeled MAb diluted 1:250 in BS with 0.1 M NaCl (BS-NaCl). Unbound antibodies were removed by washing the membranes twice in BS-NaCl and once in phosphate-buffered saline solution. In order to demonstrate non-toxin-producing P. multocida X-73 in a mixed culture, the membrane was incubated for 1 h with anti-lipopolysaccharide serum diluted 1:200 in BS-NaCl. The membrane was washed three times with BS-NaCl and incubated for 1 h with goat anti-chicken IgG diluted 1:500 in BS-NaCl. Unbound antibodies were removed by washing the membrane twice in BS-NaCl and once in phosphate-buffered saline solution. Peroxidase-labeled antibody binding to colony blots was detected with a 4-chloronaphthol-hydrogen peroxide substrate solution (Kirkegaard and Perry Laboratories). The developing reaction was stopped by washing the membranes with distilled water. Comparison of different blocking agents. In one experiment, 1% casein, 1% bovine serum albumin (Miles Scientific, Naperville, Ill.), 3% skim milk (Difco, Detroit, Mich.), and 1% fish gelatin (HiPure; Norland Products, Inc., New Brunswick, N.J.) were compared as blocking agents. Mouse lethality tests. Sterile-filtered cell extracts of sonicated bacteria were tested for lethality in BALB/c mice as described previously (22). Detection of toxin-producing P. multocida from specimen. Blood agar plates inoculated with serial 10-fold dilutions of homogenized turbinate tissues were provided by Mark R. Ackermann, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa. The tissues were from 21-day-old gnotobiotic pigs, experimentally infected at 7 days of age with P. multocida P-4533. RESULTS Detection of toxin-producing P. multocida. Colonies of bacterial cultures streaked onto an agar plate were easily lifted, and their patterns were reproduced with a single membrane. Those lifted onto nylon 66 membranes appeared more to be distinct than those lifted onto nitrocellulose membranes. All bacteria tested were readily lysed by 0.5 M NaOH within 5 min on either membrane. Comparisons of blocking agents indicated that a Tris-glycine buffer containing casein was best for eliminating nonspecific background reactions in the assay. The same buffer containing bovine serum albumin or skim milk gave acceptable results, but fish
1330
MAGYAR AND RIMLER
" 3
%
FIG. 1. Reactions, after 16 h of incubation, of toxin-producing P. multocida P-5048 (reactions 1 through 3), P-5045 (reaction 4), P-5066 (reaction 5), P-4066 (reaction 6), and P-4533B (reaction 10) and non-toxin-producing P. multocida P-5062 (reaction 7), X-73 (reaction 8), and P-4671 (reaction 9) on a nylon 66 membrane with peroxidase-labeled MAb.
gelatin was unsuitable. After colony lysis, blocking, and drying at room temperature, nylon 66 membranes could be stored in plastic bags at -70°C until they were required. Of the two media tested, blood agar was preferable to glucose starch agar because the transferred toxin-producing colonies produced more intense reactions with MAb in the membrane assay. Passage on agar did not result in any strain losing the ability to produce toxin, and no appreciable effect was detected in the reaction intensity in the membrane assay.
Figure 1 shows the membrane assay in which toxin in five P. multocida strains (P-5048, P-5045, P-5066, P-4066, and P-4533) was detected with the peroxidase-labeled MAb. The color intensities of the reactions obtained with different positive strains varied but were easily discerned. Non-toxinproducing P. multocida (P-5062, X-73, and P-4671) did not react in the assay. Specificity. Table 1 compares data for mouse lethality tests with peroxidase-labeled MAb reactions in the membrane assay. There was a complete agreement between a filtered sonic extract of P. multocida being lethal for mice and a positive colony reaction in the membrane assay. Toxin was detected in both serogroup A and D P. multocida organisms isolated from various animal sources. No relationship was found between the presence or absence of a capsule and reaction in the assay. Also, there was no correlation between serotype specificity and reaction in the assay. Control cultures of B. bronchiseptica and B. avium showed no reactions in the membrane assay, even though sterile-filtered sonic extracts of the toxin-producing Bordetella spp. were lethal for mice. The Cowan 1 strain of S. aureus, which produces protein A, showed a strong false-positive reaction in the assay.
Enumeration. The membrane assay could be used to enumerate toxin-producing P. multocida in mixed cultures
containing the organism and non-toxin-producing P. multocida or B. bronchiseptica. Well-defined toxin-producing colonies could be distinguished by the membrane assay within 12 h of incubation. However, because colonies of different strains grew at different rates, 16 to 18 h of incubation was optimum for the detection of toxin in all strains. At that time, all colonies were of sufficient size and could be easily transfered to one or more membranes. Further incubation of a toxin-producing strain for 48 h
J. CLIN. MICROBIOL.
resulted in a slight increase in the intensity of reaction in the assay. However, the specificity of the reaction with any strain did not change. Figure 2A shows a typical positive reaction in the membrane assay, in which the dark spots represent toxin-producing noncapsulated P. multocida; this membrane contained lysed colonies of a mixed culture of strain P-4533B and non-toxin-producing strain X-73. Figure 2B shows the reactions of both strains X-73 and P-4533B on the same membrane (from Fig. 2A) that was reprobed with chicken antiserotype 1 lipopolysaccharide. Comparison of Fig. 2A with Fig. 2B (see arrows) shows that colonies of toxin-producing P. multocida that were confluent with non-toxin-producing P. multocida could be distinguished by the assay. All colonies of strain P-4533, whether enumerated from pure culture or mixed cultures with Bordetella spp. or strain X-73, were distinguished by the membrane assay (data not shown). Detection of toxin-producing P. multocida in specimens. Strain P-4533 was reisolated in pure culture from turbinate specimens from experimentally infected gnotobiotic pigs. All colonies on primary isolation blood agar plates transfered to nylon 66 membranes produced a strong positive reaction in the assay with peroxidase-labeled MAb. DISCUSSION The P. multocida strains examined in this study represent a wide range of hosts, geographic origins, capsule serogroups, and somatic serotypes. Toxin-producing P. multocida organisms were found among noncapsulated and serogroup A and D organisms as well as among organisms with different somatic serotypes. Positive reactions in the membrane assay among these strains correlated completely with their lethal toxicities for mice. These findings are similar to those of previous reports in which P. multocida toxins from organisms with different serotypes were found to be antigenically similar or identical (9, 22). No relationship was detected between epitopes of the B. bronchiseptica or B. avium toxins and the toxin of P. multocida by the membrane assay. Atrophic rhinitis of swine is a disease in which detection of toxin-producing P. multocida is essential for diagnosis and control. The membrane colony-blot assay reported here is a highly specific test with the potential for detecting this organism in primary isolations from clinical material. In animals with atrophic rhinitis, toxin-producing P. multocida is often isolated from mixed cultures by chance, especially because it can occur together with non-toxin-producing P. multocida (unpublished data). The fact that a large number of colonies can be examined from a single isolation plate improves the probability of detecting toxin-producing P. multocida and making a presumptive diagnosis of atrophic rhinitis. In instances when specimens with a resident flora are examined, it may be necessary to culture the specimens on selective medium to detect P. multocida. Several selective media for isolation of this organism are available (23). Use of a selective medium would be especially important to avoid the growth of staphylococci and certain streptococci. These
gram-positive organisms can produce false-positive reactions by nonimmunological binding of the Fc component of peroxidase-labeled MAb. The different selective media use growth inhibitors such as potassium tellurite, chloral hydrate, dyes, and antibiotics. We have not yet evaluated the influence of different selective media or their constituents on toxin production by P. multocida colonies. However, anticipating that this may be a problem, preliminary experiments
VOL. 29, 1991
TOXIN-PRODUCING P. MULTOCIDA
1331
FIG. 2. (A) Peroxidase-labeled MAb detection of toxin-producing strain P-4533B colonies grown for 16 h on blood agar and transfered to a nylon 66 membrane; P-4533B colonies were in a mixed culture containing non-toxin-producing strain X-73. (B) Demonstration of P-4533B and X-73 mixed culture colonies after reprobing of the membrane in panel A with anti-lipopolysaccharide serum. Arrows indicate confluent colonies of P-4533B and X-73 in which only P-4533B was detected in panel A.
were done with disposable replicate platers (Accutran; Schleicher & Schuell). The findings suggest that colonies can be easily replicated from a selective medium onto blood agar medium for enumeration of toxin-producing strains by the membrane assay. The peroxidase-labeled MAb in the membrane assay clearly differentiates toxin-producing P. multocida from other isolates. The procedure used for the assay does not require elaborate equipment, tissue culture, or animals, and the preparation of samples is very simple. Because the test is rapid, not very expensive, and suitable for examination of large numbers of isolated strains within a few hours, it is suitable for most diagnostic laboratories. The test should be useful for epidemiologic surveys among animal populations infected with P. multocida as well as for the diagnosis of atrophic rhinitis. REFERENCES 1. Baalsrud, K. J. 1987. Atrophic rhinitis in goats in Norway. Vet. Rec. 121:350-353. 2. Chanter, N., T. Magyar, and J. M. Rutter. 1989. Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Res. Vet. Sci. 47:48-53. 3. Chanter, N., J. M. Rutter, and P. D. Luther. 1986. Rapid detection of toxigenic Pasteurella multocida by an agar overlay method. Vet. Rec. 119:629-630. 4. Chanter, N., J. M. Rutter, and A. MacKenzie. 1986. Partial purification of an osteolytic toxin from Pasteurella multocida. J. Gen. Microbiol. 132:1089-1097. 5. de Jong, M. F. 1983. Atrophic rhinitis caused by intranasal or intramuscular administration of broth culture and broth culture filtrates containing AR toxin of Pasteurella multocida, p. 136146. In K. B. Pederson and N. C. Nielsen (ed.), Atrophic rhinitis of pigs. Commission of the European Communities, Brussels. 6. de Jong, M. F., H. L. Oei, and G. J. Tetenburg. 1980. AR-
7.
8.
9.
10. 11.
12.
13. 14.
15. 16. 17.
pathogenicity tests for Pasteurella multocida isolates. Proc. Int. Pig Vet. Soc. Congr., p. 211, Copenhagen, Denmark. Dominick, M. A., and R. B. Rimler. 1986. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am. J. Vet. Res. 47:1532-1536. Elling, F., and K. B. Pedersen. 1983. Atrophic rhinitis in pigs induced by a dermonecrotic type A strain of Pasteurella multocida, p. 123-135. In K. B. Pederson and N. C. Nielsen (ed.), Atrophic rhinitis of pigs. Commission of the European Communities, Brussels. Foged, N. T., J. P. Nielsen, and K. B. Pedersen. 1988. Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 26:1419-1420. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis. 16:925-936. Il'ina, Z. M., and I. Zasukhin. 1975. Role of pasteurella toxins in the pathogenesis of infectious atrophic rhinitis. Sb. Nauchn. Rab. Sib. Nauch. Vet. Inst. Omsk 25:76-86. (In Russian.) Kielstein, P. 1986. On the occurrence of toxin-producing Pasteurella multocida strains in atrophic rhinitis and pneumonias of swine and cattle. J. Vet. Med. 33:418-424. Magyar, T. 1989. Study of the toxin-producing ability of Pasteurella multocida in mice. Acta Vet. Hung. 37:319-325. Martineau-Doize, B., J. C. Frantz, and G. Martineau. 1990. Effects of purified Pasteurella multocida dermonecrotoxin on cartilage and bone of the nasal conchae of the piglet. Anat. Rec. 228:237-246. Nakai, T., A. Sawata, M. Tsuji, and K. Kume. 1984. Characterization of dermonecrotic toxin produced by serotype D strains of Pasteurella multocida. Am. J. Vet. Res. 45:2410-2413. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labeled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. Nielson, J. P., M. Bisgaard, and K. B. Pederson. 1986. Produc-
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22.
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MAGYAR AND RIMLER
tion of toxin in strains previously classified as Pasteurella multocida. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 94:203-204. Pederson, K. B., and K. Barfod. 1981. The aetiological significance ofBordetella bronchiseptica and Pasteurella multocida in atrophic rhinitis of swine. Nord. Vet. 33:513-522. Pijoan, C., A. Lastra, C. Ramirez, and A. Leman. 1984. Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine. Am. J. Vet. Res. 185:522-523. Rhoades, K. R., and R. B. Rimler. 1990. Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts. Avian Dis. 34:384-388. Rimler, R. B., R. D. Angus, and M. Phillips. 1989. Evaluation of the specificity of Pasteurella multocida somatic antigen-typing antisera prepared in chickens, using ribosome-lipopolysaccharide complexes as inocula. Am. J. Vet. Res. 50:29-31. Rimler, R. B., and K. A. Brogden. 1986. Pasteurella multocida
23. 24.
25. 26. 27.
isolated from rabbits and swine: serologic types and toxin production. Am. J. Vet. Res. 47:730-737. Rimler, R. B., and K. R. Rhoades. 1989. Pasteurella multocida, p. 35-73. In C. Adlam and J. M. Rutter (ed.), Pasteurella and pasteurellosis. Academic Press, Inc., San Diego, Calif. Rutter, J. M. 1983. Virulence of Pasteurella multocida in atrophic rhinitis of gnotobiotic pigs infected with Bordetella bronchiseptica. Res. Vet. Sci. 34:287-295. Rutter, J. M., and P. D. Luther. 1984. Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs. Vet. Rec. 114:393-396. Rutter, J. M., and A. MacKenzie. 1984. Pathogenesis of atrophic rhinitis in pigs: a new perspective. Vet. Rec. 114:89-90. Rutter, J. M., and X. Rojas. 1982. Atrophic rhinitis in gnotobiotic piglets: differences in the pathogenicity of Pasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec. 110:531-535.
Vol. 29, No. 7
Detection and Enumeration of Toxin-Producing Pasteurella multocida with a Colony-Blot Assay TIBOR MAGYAR' AND RICHARD B. RIMLER2* Veterinary Medical Research Institute, Hungarian Academy of Sciences, H-1581 Budapest, Hungary,' and Avian Diseases Research Unit, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, P.O. Box 70, Ames, Iowa 500102 Received 31 December 1990/Accepted 2 April 1991
Colonies of toxin-producing Pasteurella multocida were detected with peroxidase-labeled monoclonal antibodies by a membrane assay. Examination of the specificity of the assay with 29 P. multocida cultures representing various geographic origins, hosts, and serotypes indicated that the test was specific for toxinproducing strains. No cross-reactions were observed with Bordetella species that can be associated with P. multocida in producing diseases in animals. A single membrane could be used to assay several isolated strains for toxin production or to enumerate toxin-producing colonies in mixed cultures. Toxin-producing P. multocida colonies were detected in primary cultures; hence, the assay appears to have good potential for widespread application with clinical samples. In the present report we describe an inexpensive and simple membrane assay with peroxidase-labeled MAb to recognize and enumerate toxin-producing P. multocida.
Progressive atrophic rhinitis of swine is a complex disease in which toxin-producing serogroup D Pasteurella multocida together with toxin-producing Bordetella bronchiseptica are recognized as the major etiologic agents (2, 18, 26). Although the dynamics of the interaction between these two agents in producing the disease are not yet understood, inoculation of pigs with cell extracts of toxin-producing P. multocida alone, either intranasally (11) or intraperitoneally (27), has reproduced the characteristic lesions of severe turbinate atrophy. These findings indicated that a toxin plays a central role in the disease. This role was confirmed when the characteristic lesions developed after intranasal (7) or intraperitoneal (4) inoculation of gnotobiotic pigs with protein toxin purified from serogroup D P. multocida. Although most reports describe toxin-producing serogroup D P. multocida, toxin-producing serogroup A organisms have also been isolated from swine (5, 8, 19), and the toxin from these organisms is either antigenically similar or identical to that from serogroup D organisms (9, 22). Besides affecting swine, recent research indicates that toxin-producing P. multocida organisms are involved in the etiology of naturally occurring progressive atrophic rhinitis of goats (1). Toxin-producing P. multocida has been isolated from rabbits, cattle, cats, dogs, and turkeys (6, 12, 17, 20, 22). However, the role of P. multocida toxin in species other than swine and goats has not been clarified. P. multocida toxin can be detected in cell extracts and culture filtrates by its lethality and lienotoxicity for mice (13, 15, 24) and production of dermonecrosis in guinea pig skin (5). Recognition of toxin-producing organisms by these methods is time-consuming, and the number of samples that can be processed is limited. In vitro techniques such as cytopathogenic effects in tissue culture (25), the agar overlay method (3), and an enzyme-linked immunosorbent assay with toxin-specific monoclonal antibodies (MAbs) (9) can recognize toxin-producing P. multocida. While these in vitro techniques allow testing of a larger number of suspect toxin-producing P. multocida isolates, they are cumbersome and require expensive equipment. *
MATERIALS AND METHODS
Bacteria. A total of 29 P. multocida strains representing various hosts, geographic origins, and serotypes were examined (Table 1). Typing of capsule serogroup antigen was done by passive hemagglutination tests as described previously (22). Somatic serotypes of these strains were determined by the gel diffusion precipitin test described by Heddleston et al. (10). B. bronchiseptica P-4609 and P-4607, a toxin-producing and a non-toxin-producing strain, respectively, from pigs affected by atrophic rhinitis (in Iowa), and Bordetella avium P-4148, a toxin-producing isolate from a turkey in Ohio, were used as control cultures. For examination of the potential of nonimmunological immunoglobulin G (IgG)-binding bacteria in producing falsepositive reactions, the Cowan 1 strain of Staphylococcus aureus (ATCC 12598) was included in the study. Preparation of samples. Bacterial strains were cultured on glucose starch agar plates and 5% bovine blood agar plates at 37°C. Colonies were examined in the membrane assay after 12, 16 to 18, and 48 h of growth. For enumeration experiments, growth was suspended in tryptose broth, and the suspension was adjusted to a density equal to a McFarland no. 1 nephelometer standard. Serial dilutions of the suspensions were made in tryptose broth and plated onto blood agar to result in an optimum of approximately 80 to 100 colonies per plate. Strains were tested in pure culture, except when mixed cultures of toxin-producing and non-toxin-producing P. multocida or B. bronchiseptica were prepared to examine the specificity of the assay. For examination of several individual strains, 10 or more cultures were streaked as separate 2-cm lines on the same agar plate. Antibodies. Mouse ascitic fluid containing IgG MAbs specific to the toxin of serogroup D P. multocida was made with hybridoma ot-DNT-1B2A3 and provided by lone Stoll, National Veterinary Services Laboratories, Science and
Corresponding author. 1328
TOXIN-PRODUCING P. MULTOCIDA
VOL. 29, 1991
TABLE 1. Lethal toxicities of filtered sonic extracts and colony reactions in a membrane assay of selected P. multocida isolates representing different hosts, origins, and serotypes Strain
P-4066 P-4067 P-3766 P-4671 P-4672 P-4673 P-5049 P-5050 P-5062 P-5066 P-5069 P-5070
Host
Geographic ongin
Rabbit Belgium Rabbit Belgium Rabbit Belgium Rabbit Michigan Rabbit Michigan Rabbit Michigan Swine Norway Swine Norway Swine Norway Swine Norway Swine Norway Swine Norway P-%1 Swine Maryland P-3018 Swine Brazil P-4531 Swine Sweden P-45331 Swine Sweden P-4533B Swine Sweden P-4976 Swine Norway P-4275 Swine Arkansas P-4980 Goat Norway P-5041 Goat Norway P-5042 Goat Norway P-5043 Goat Norway P-5044 Goat Norway P-5045 Goat Norway P-5046 Goat Norway P-5048 Goat Norway P-4123 Cattle Mali X-73 Chicken Maryland a MA, membrane assay.
Serotype
D:3, 4" D:3, 12 D: 12 D:3 D:3 D: 12 A:3 A:3 A:3, 4 A:3 A:3 A:3 D:3 D: 12 D:12 D:3 -:3c A:3 -:12
D:3 D:3, 4 D:3, D:3, D:3, D:3, D:4, A:3 E:5 A: 1
4, 12 4, 12 4 4 7, 12
Lethality for Reaction in mice MA
+ +
+ +
-
-
-
-
-
-
-
-
-
-
-
-
+ +
+ +
+
+
+ + + + + + + + + + + + +
+ + + + + + + + + + + + +
-
-
b The letter indicates capsule serogroup; the number(s) indicates reaction with serotype antiserum. c -, absence of a letter for the capsule group denotes a noncapsulated strain.
Technology, Animal and Plant Health Inspection Service, U.S. Department of Agriculture, Ames, Iowa. Hybridoma a-DNT-1B2A3 (to be deposited with the American Type Culture Collection) was prepared with mice immunized with a toxoid. The toxoid, a gift from J. C. Frantz, Norden Laboratories, Inc., Lincoln, Nebr., was made by a proprietary procedure with toxin purified from the NOR-1 strain of P. multocida, as described by Martineau-Doize et al. (14). To purify MAb, the ascitic fluid was mixed 1.5:1 with Seroclear (Calbiochem, La Jolla, Calif.) and centrifuged. The supernatant was removed by aspiration, diluted with 4 volumes of Tris-NaCl buffer (0.05 M Tris, 0.15 M NaCI [pH 7.3]), and applied to a column (1.6 by 14 cm) of Avid Al gel (BioProbe, Tustin, Calif.) equilibrated with the same buffer. IgG MAb was eluted from the column with 3 M KSCN in Tris-NaCl buffer, dialyzed against Tris-NaCl buffer, and concentrated over an XM-50 membrane (Amicon Corp., Danvers, Mass.). The purified MAbs were labeled with horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) by the method of Nakane and Kawaoi (16). A block titration in a dot blot assay was done with purified P. multocida toxin (22) versus peroxidase-labeled MAb. The detection limit of a 1:250 working dilution of labeled MAb was 0.042 ag of purified toxin. Chicken antiserum specific for the lipopolysaccharide of P. multocida serotype 1 was prepared as described previously (21). Peroxidase-labeled goat anti-chicken IgG was
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from Kirkegaard and Perry Laboratories (Gaithersburg, Md.). Membrane assay. Modified nylon 66 (Nytran; Schleicher & Schuell, Keane, N.H.) or nitrocellulose (BA 85; Schleicher & Schuell) membrane circles were used for the assay. For colony transfers, the dry membranes were gently placed onto an agar surface, pressed lightly and evenly with an L-shaped glass rod, removed with forceps, and placed (colony side up) onto blotting paper saturated with 0.5 M NaOH. The colonies were allowed to lyse for about 5 min. After lysis, the membranes were transferred to blotting paper saturated with phosphate-buffered saline solution (pH 7.0) for neutralization. All steps thereafter were done in petri plates at room temperature with constant horizontal rotation at about 80 rpm. Nonreacted binding sites on the membranes were blocked by incubating them for 1 h in a solution (BS) that contained 1% casein (Fisher Chemical Co., Fair Lawn, N.J.), 0.05% Tween 20, 0.025 M Tris, and 0.192 M glycine in distilled water (pH 8.3). After blocking, membranes were incubated for 1 h with peroxidase-labeled MAb diluted 1:250 in BS with 0.1 M NaCl (BS-NaCl). Unbound antibodies were removed by washing the membranes twice in BS-NaCl and once in phosphate-buffered saline solution. In order to demonstrate non-toxin-producing P. multocida X-73 in a mixed culture, the membrane was incubated for 1 h with anti-lipopolysaccharide serum diluted 1:200 in BS-NaCl. The membrane was washed three times with BS-NaCl and incubated for 1 h with goat anti-chicken IgG diluted 1:500 in BS-NaCl. Unbound antibodies were removed by washing the membrane twice in BS-NaCl and once in phosphate-buffered saline solution. Peroxidase-labeled antibody binding to colony blots was detected with a 4-chloronaphthol-hydrogen peroxide substrate solution (Kirkegaard and Perry Laboratories). The developing reaction was stopped by washing the membranes with distilled water. Comparison of different blocking agents. In one experiment, 1% casein, 1% bovine serum albumin (Miles Scientific, Naperville, Ill.), 3% skim milk (Difco, Detroit, Mich.), and 1% fish gelatin (HiPure; Norland Products, Inc., New Brunswick, N.J.) were compared as blocking agents. Mouse lethality tests. Sterile-filtered cell extracts of sonicated bacteria were tested for lethality in BALB/c mice as described previously (22). Detection of toxin-producing P. multocida from specimen. Blood agar plates inoculated with serial 10-fold dilutions of homogenized turbinate tissues were provided by Mark R. Ackermann, National Animal Disease Center, Agricultural Research Service, U.S. Department of Agriculture, Ames, Iowa. The tissues were from 21-day-old gnotobiotic pigs, experimentally infected at 7 days of age with P. multocida P-4533. RESULTS Detection of toxin-producing P. multocida. Colonies of bacterial cultures streaked onto an agar plate were easily lifted, and their patterns were reproduced with a single membrane. Those lifted onto nylon 66 membranes appeared more to be distinct than those lifted onto nitrocellulose membranes. All bacteria tested were readily lysed by 0.5 M NaOH within 5 min on either membrane. Comparisons of blocking agents indicated that a Tris-glycine buffer containing casein was best for eliminating nonspecific background reactions in the assay. The same buffer containing bovine serum albumin or skim milk gave acceptable results, but fish
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MAGYAR AND RIMLER
" 3
%
FIG. 1. Reactions, after 16 h of incubation, of toxin-producing P. multocida P-5048 (reactions 1 through 3), P-5045 (reaction 4), P-5066 (reaction 5), P-4066 (reaction 6), and P-4533B (reaction 10) and non-toxin-producing P. multocida P-5062 (reaction 7), X-73 (reaction 8), and P-4671 (reaction 9) on a nylon 66 membrane with peroxidase-labeled MAb.
gelatin was unsuitable. After colony lysis, blocking, and drying at room temperature, nylon 66 membranes could be stored in plastic bags at -70°C until they were required. Of the two media tested, blood agar was preferable to glucose starch agar because the transferred toxin-producing colonies produced more intense reactions with MAb in the membrane assay. Passage on agar did not result in any strain losing the ability to produce toxin, and no appreciable effect was detected in the reaction intensity in the membrane assay.
Figure 1 shows the membrane assay in which toxin in five P. multocida strains (P-5048, P-5045, P-5066, P-4066, and P-4533) was detected with the peroxidase-labeled MAb. The color intensities of the reactions obtained with different positive strains varied but were easily discerned. Non-toxinproducing P. multocida (P-5062, X-73, and P-4671) did not react in the assay. Specificity. Table 1 compares data for mouse lethality tests with peroxidase-labeled MAb reactions in the membrane assay. There was a complete agreement between a filtered sonic extract of P. multocida being lethal for mice and a positive colony reaction in the membrane assay. Toxin was detected in both serogroup A and D P. multocida organisms isolated from various animal sources. No relationship was found between the presence or absence of a capsule and reaction in the assay. Also, there was no correlation between serotype specificity and reaction in the assay. Control cultures of B. bronchiseptica and B. avium showed no reactions in the membrane assay, even though sterile-filtered sonic extracts of the toxin-producing Bordetella spp. were lethal for mice. The Cowan 1 strain of S. aureus, which produces protein A, showed a strong false-positive reaction in the assay.
Enumeration. The membrane assay could be used to enumerate toxin-producing P. multocida in mixed cultures
containing the organism and non-toxin-producing P. multocida or B. bronchiseptica. Well-defined toxin-producing colonies could be distinguished by the membrane assay within 12 h of incubation. However, because colonies of different strains grew at different rates, 16 to 18 h of incubation was optimum for the detection of toxin in all strains. At that time, all colonies were of sufficient size and could be easily transfered to one or more membranes. Further incubation of a toxin-producing strain for 48 h
J. CLIN. MICROBIOL.
resulted in a slight increase in the intensity of reaction in the assay. However, the specificity of the reaction with any strain did not change. Figure 2A shows a typical positive reaction in the membrane assay, in which the dark spots represent toxin-producing noncapsulated P. multocida; this membrane contained lysed colonies of a mixed culture of strain P-4533B and non-toxin-producing strain X-73. Figure 2B shows the reactions of both strains X-73 and P-4533B on the same membrane (from Fig. 2A) that was reprobed with chicken antiserotype 1 lipopolysaccharide. Comparison of Fig. 2A with Fig. 2B (see arrows) shows that colonies of toxin-producing P. multocida that were confluent with non-toxin-producing P. multocida could be distinguished by the assay. All colonies of strain P-4533, whether enumerated from pure culture or mixed cultures with Bordetella spp. or strain X-73, were distinguished by the membrane assay (data not shown). Detection of toxin-producing P. multocida in specimens. Strain P-4533 was reisolated in pure culture from turbinate specimens from experimentally infected gnotobiotic pigs. All colonies on primary isolation blood agar plates transfered to nylon 66 membranes produced a strong positive reaction in the assay with peroxidase-labeled MAb. DISCUSSION The P. multocida strains examined in this study represent a wide range of hosts, geographic origins, capsule serogroups, and somatic serotypes. Toxin-producing P. multocida organisms were found among noncapsulated and serogroup A and D organisms as well as among organisms with different somatic serotypes. Positive reactions in the membrane assay among these strains correlated completely with their lethal toxicities for mice. These findings are similar to those of previous reports in which P. multocida toxins from organisms with different serotypes were found to be antigenically similar or identical (9, 22). No relationship was detected between epitopes of the B. bronchiseptica or B. avium toxins and the toxin of P. multocida by the membrane assay. Atrophic rhinitis of swine is a disease in which detection of toxin-producing P. multocida is essential for diagnosis and control. The membrane colony-blot assay reported here is a highly specific test with the potential for detecting this organism in primary isolations from clinical material. In animals with atrophic rhinitis, toxin-producing P. multocida is often isolated from mixed cultures by chance, especially because it can occur together with non-toxin-producing P. multocida (unpublished data). The fact that a large number of colonies can be examined from a single isolation plate improves the probability of detecting toxin-producing P. multocida and making a presumptive diagnosis of atrophic rhinitis. In instances when specimens with a resident flora are examined, it may be necessary to culture the specimens on selective medium to detect P. multocida. Several selective media for isolation of this organism are available (23). Use of a selective medium would be especially important to avoid the growth of staphylococci and certain streptococci. These
gram-positive organisms can produce false-positive reactions by nonimmunological binding of the Fc component of peroxidase-labeled MAb. The different selective media use growth inhibitors such as potassium tellurite, chloral hydrate, dyes, and antibiotics. We have not yet evaluated the influence of different selective media or their constituents on toxin production by P. multocida colonies. However, anticipating that this may be a problem, preliminary experiments
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TOXIN-PRODUCING P. MULTOCIDA
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FIG. 2. (A) Peroxidase-labeled MAb detection of toxin-producing strain P-4533B colonies grown for 16 h on blood agar and transfered to a nylon 66 membrane; P-4533B colonies were in a mixed culture containing non-toxin-producing strain X-73. (B) Demonstration of P-4533B and X-73 mixed culture colonies after reprobing of the membrane in panel A with anti-lipopolysaccharide serum. Arrows indicate confluent colonies of P-4533B and X-73 in which only P-4533B was detected in panel A.
were done with disposable replicate platers (Accutran; Schleicher & Schuell). The findings suggest that colonies can be easily replicated from a selective medium onto blood agar medium for enumeration of toxin-producing strains by the membrane assay. The peroxidase-labeled MAb in the membrane assay clearly differentiates toxin-producing P. multocida from other isolates. The procedure used for the assay does not require elaborate equipment, tissue culture, or animals, and the preparation of samples is very simple. Because the test is rapid, not very expensive, and suitable for examination of large numbers of isolated strains within a few hours, it is suitable for most diagnostic laboratories. The test should be useful for epidemiologic surveys among animal populations infected with P. multocida as well as for the diagnosis of atrophic rhinitis. REFERENCES 1. Baalsrud, K. J. 1987. Atrophic rhinitis in goats in Norway. Vet. Rec. 121:350-353. 2. Chanter, N., T. Magyar, and J. M. Rutter. 1989. Interactions between Bordetella bronchiseptica and toxigenic Pasteurella multocida in atrophic rhinitis of pigs. Res. Vet. Sci. 47:48-53. 3. Chanter, N., J. M. Rutter, and P. D. Luther. 1986. Rapid detection of toxigenic Pasteurella multocida by an agar overlay method. Vet. Rec. 119:629-630. 4. Chanter, N., J. M. Rutter, and A. MacKenzie. 1986. Partial purification of an osteolytic toxin from Pasteurella multocida. J. Gen. Microbiol. 132:1089-1097. 5. de Jong, M. F. 1983. Atrophic rhinitis caused by intranasal or intramuscular administration of broth culture and broth culture filtrates containing AR toxin of Pasteurella multocida, p. 136146. In K. B. Pederson and N. C. Nielsen (ed.), Atrophic rhinitis of pigs. Commission of the European Communities, Brussels. 6. de Jong, M. F., H. L. Oei, and G. J. Tetenburg. 1980. AR-
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pathogenicity tests for Pasteurella multocida isolates. Proc. Int. Pig Vet. Soc. Congr., p. 211, Copenhagen, Denmark. Dominick, M. A., and R. B. Rimler. 1986. Turbinate atrophy in gnotobiotic pigs intranasally inoculated with protein toxin isolated from type D Pasteurella multocida. Am. J. Vet. Res. 47:1532-1536. Elling, F., and K. B. Pedersen. 1983. Atrophic rhinitis in pigs induced by a dermonecrotic type A strain of Pasteurella multocida, p. 123-135. In K. B. Pederson and N. C. Nielsen (ed.), Atrophic rhinitis of pigs. Commission of the European Communities, Brussels. Foged, N. T., J. P. Nielsen, and K. B. Pedersen. 1988. Differentiation of toxigenic from nontoxigenic isolates of Pasteurella multocida by enzyme-linked immunosorbent assay. J. Clin. Microbiol. 26:1419-1420. Heddleston, K. L., J. E. Gallagher, and P. A. Rebers. 1972. Fowl cholera: gel diffusion precipitin test for serotyping Pasteurella multocida from avian species. Avian Dis. 16:925-936. Il'ina, Z. M., and I. Zasukhin. 1975. Role of pasteurella toxins in the pathogenesis of infectious atrophic rhinitis. Sb. Nauchn. Rab. Sib. Nauch. Vet. Inst. Omsk 25:76-86. (In Russian.) Kielstein, P. 1986. On the occurrence of toxin-producing Pasteurella multocida strains in atrophic rhinitis and pneumonias of swine and cattle. J. Vet. Med. 33:418-424. Magyar, T. 1989. Study of the toxin-producing ability of Pasteurella multocida in mice. Acta Vet. Hung. 37:319-325. Martineau-Doize, B., J. C. Frantz, and G. Martineau. 1990. Effects of purified Pasteurella multocida dermonecrotoxin on cartilage and bone of the nasal conchae of the piglet. Anat. Rec. 228:237-246. Nakai, T., A. Sawata, M. Tsuji, and K. Kume. 1984. Characterization of dermonecrotic toxin produced by serotype D strains of Pasteurella multocida. Am. J. Vet. Res. 45:2410-2413. Nakane, P. K., and A. Kawaoi. 1974. Peroxidase-labeled antibody: a new method of conjugation. J. Histochem. Cytochem. 22:1084-1091. Nielson, J. P., M. Bisgaard, and K. B. Pederson. 1986. Produc-
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tion of toxin in strains previously classified as Pasteurella multocida. Acta Pathol. Microbiol. Immunol. Scand. Sect. B 94:203-204. Pederson, K. B., and K. Barfod. 1981. The aetiological significance ofBordetella bronchiseptica and Pasteurella multocida in atrophic rhinitis of swine. Nord. Vet. 33:513-522. Pijoan, C., A. Lastra, C. Ramirez, and A. Leman. 1984. Isolation of toxigenic strains of Pasteurella multocida from lungs of pneumonic swine. Am. J. Vet. Res. 185:522-523. Rhoades, K. R., and R. B. Rimler. 1990. Virulence and toxigenicity of capsular serogroup D Pasteurella multocida strains isolated from avian hosts. Avian Dis. 34:384-388. Rimler, R. B., R. D. Angus, and M. Phillips. 1989. Evaluation of the specificity of Pasteurella multocida somatic antigen-typing antisera prepared in chickens, using ribosome-lipopolysaccharide complexes as inocula. Am. J. Vet. Res. 50:29-31. Rimler, R. B., and K. A. Brogden. 1986. Pasteurella multocida
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isolated from rabbits and swine: serologic types and toxin production. Am. J. Vet. Res. 47:730-737. Rimler, R. B., and K. R. Rhoades. 1989. Pasteurella multocida, p. 35-73. In C. Adlam and J. M. Rutter (ed.), Pasteurella and pasteurellosis. Academic Press, Inc., San Diego, Calif. Rutter, J. M. 1983. Virulence of Pasteurella multocida in atrophic rhinitis of gnotobiotic pigs infected with Bordetella bronchiseptica. Res. Vet. Sci. 34:287-295. Rutter, J. M., and P. D. Luther. 1984. Cell culture assay for toxigenic Pasteurella multocida from atrophic rhinitis of pigs. Vet. Rec. 114:393-396. Rutter, J. M., and A. MacKenzie. 1984. Pathogenesis of atrophic rhinitis in pigs: a new perspective. Vet. Rec. 114:89-90. Rutter, J. M., and X. Rojas. 1982. Atrophic rhinitis in gnotobiotic piglets: differences in the pathogenicity of Pasteurella multocida in combined infections with Bordetella bronchiseptica. Vet. Rec. 110:531-535.
