Vol. 28, No. 1
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 103-107
0095-1137/90/010103-05$02.00/0 Copyright © 1990, American Society for Microbiology
Development and Epidemiological Applications of a Bacteriophage Typing System for Typing Pasteurella multocida JENS PETER NIELSENl* AND VIBEKE THAMDRUP ROSDAHL2 National Veterinary Laboratory' and Staphylococcus Laboratory, State Serum Institute,2 Copenhagen, Denmark Received 26 June 1989/Accepted 20 September 1989
A bacteriophage typing system was developed for typing toxigenic and nontoxigenic strains of Pasteurella multocida. A phage set of 24 phages with different lytic spectra was isolated after mitomycin treatment of P. multocida strains, isolated mainly from pigs from herds with atrophic rhinitis. On a test set of 97 different strains isolated from pigs, these 24 phages were able to type 87% of the strains. The 97 test strains could be subdivided into 31 different types by reaction with the 24 phages. The reproducibility after subculture and storage of the strains was good (95%). Phage typing of 217 toxigenic P. multocida field isolates from 37 pig herds predominately with clinically atrophied rhinitis resulted in 18 different phage types and an overall typability of 68%. Of 24 herds from which more than three isolates of toxigenic P. multocida were obtained, a single phage type was demontrated in 5 herds, while in 9 herds a single phage type represented at least half of the isolates. The phage types in the remaining 10 herds revealed no dominating phage type. The phage typing system described appears to be a valuable epidemiological tool for studying the spread of P. multocida.
Pasteurella multocida is the cause of disease in a variety of mammalian and avian species. In recent years, toxigenic P. multocida subsp. multocida has been recognized as the cause of progressive atrophic rhinitis in pigs (13). The epidemiology and spread of this organism are, however, not well understood, mainly due to lack of a suitable typing
isolated from 73 pig herds. The strains originated from specific-pathogen-free herds freé of progressive atrophic rhinitis as well as from conventional herds. Capsular types and toxin production are shown in Table 1. Furthermore, 388 strains were examined for phage production. The strains were mainly isolated from pigs, but 1 originated from a cat and 53 were of human origin. Field isolates. Isolates of toxigenic P. multocida were obtained from 37 Danish sow herds. Nasal swabs from 10 4-week-old and 10 8-week-old pigs were spread on selective blood agar plates (1), followed by subcultivation of P. multocida-like colonies. Subcultures were identified and characterized as described below. Identification of strains. P. multocida subsp. multocida strains were identified as described by Foged et al. (7). Toxin production. P. multocida strains were tested for toxin production in a cell culture assay as described by Rutter and Luther (15). Capsular type. Nonserological methods (4, 5) were used to determine capsular type A or D. Media. Subcultivation was performed either on 5% blood agar plates or in tryptic broth. Phage typing was investigated on extract agar plates with CaCl2 (2) or Levinthal agar prepared by adding 100 ml of defibrinated horse blood to 1,200 ml of Difco brain heart infusion agar. After filtration at 80°C, 15 ml of glucose (33%), 10 ml of yeast extract (50%), 4 ml of NAD solution (5%), and 2 ml of CaCl2 2H20 (10%) were added at 45°C. This agar was prepared with or without CaC12. Chemicals. Mitomycin C was obtained from Sigma Chemical Co. and dissolved in saline (10 ,xg/ml). Hyaluronidase (lyophilized from bovine testis) was also from Sigma (batch H-3506; 295 NF units/mg of solid), and a 500-U/ml solution in saline was used. Phage release by mitomycin treatment (reverse typing). Phage induction was performed as described by de Saxe and Notley (6). An overnight broth culture of the strain was diluted 1/100 in fresh tryptic broth and shaken for 2 h at 37°C. To 3.8 ml of the broth culture was added 0.2 ml of mitomycin solution (10 ,ug/ml), and incubation was continued for 30 min. After centrifugation, the bacterial pellet was suspended
system.
Serotyping based on capsular antigen has been used for several years (3, 14), but strains associated with progressive atrophic rhinitis belong to capsular type A or D (7); thus, no sufficient discrimination can be obtained. Also, serotyping based on somatic antigens has been of limited value for epidemiological studies of progressive atrophic rhinitis (14). Investigations on cell envelope proteins and lipopolysaccharides have revealed only a limited number of P. multocida clones (9). The occurrence of bacteriophages in P. multocida has been reported earlier (8). Phage typing has been widely used for typing salmonella, staphylococci, and pseudomonads (12). Phage typing is based on differences in the ability of a number of different phages to lyse, or not lyse, bacterial strains. The purpose of this study was to develop a P. multocida phage typing system which could fulfill the requirements of good discriminatory power, high typability, and good reproducibility. Such a system would be useful in epidemiological studies of the spread of P. multocida both within and between herds and thereby identify sources of infection in pig herds previously free of toxigenic P. multocida. Furthermore, the system might be used as a typing system in infections caused by nontoxigenic P. multocida in other mammalian and avian species.
-
MATERIALS AND METHODS Strains. To develop the phage set, a total of 485 strains of P. multocida subsp. multocida were examined (10). Lytic phages (plaques) were demonstrated on a test set of 97 of these strains of P. multocida, consisting of 3 laboratory strains (NCTC 12177, 4158, and NCTC 12178) and 94 strains *
Corresponding author. 103
J. CLIN. MICROBIOL.
NIELSEN AND ROSDAHL
104
stage, plate cultures could be treated with chloroform vapors for 0.5 h to kill bacterial growth without inactivating the phages. After centrifugation and filtration, 10-fold dilutions of the phage suspension were prepared and 0.02 ml of each dilution was placed on a plate, flooded with the propagating strain. After incubation and determination of PFU per milliliter, we calculated the RTD (i.e., the dilution which contains in 1 drop enough phages for nearly confluent lysis). If the phage suspension was
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1990, p. 103-107
0095-1137/90/010103-05$02.00/0 Copyright © 1990, American Society for Microbiology
Development and Epidemiological Applications of a Bacteriophage Typing System for Typing Pasteurella multocida JENS PETER NIELSENl* AND VIBEKE THAMDRUP ROSDAHL2 National Veterinary Laboratory' and Staphylococcus Laboratory, State Serum Institute,2 Copenhagen, Denmark Received 26 June 1989/Accepted 20 September 1989
A bacteriophage typing system was developed for typing toxigenic and nontoxigenic strains of Pasteurella multocida. A phage set of 24 phages with different lytic spectra was isolated after mitomycin treatment of P. multocida strains, isolated mainly from pigs from herds with atrophic rhinitis. On a test set of 97 different strains isolated from pigs, these 24 phages were able to type 87% of the strains. The 97 test strains could be subdivided into 31 different types by reaction with the 24 phages. The reproducibility after subculture and storage of the strains was good (95%). Phage typing of 217 toxigenic P. multocida field isolates from 37 pig herds predominately with clinically atrophied rhinitis resulted in 18 different phage types and an overall typability of 68%. Of 24 herds from which more than three isolates of toxigenic P. multocida were obtained, a single phage type was demontrated in 5 herds, while in 9 herds a single phage type represented at least half of the isolates. The phage types in the remaining 10 herds revealed no dominating phage type. The phage typing system described appears to be a valuable epidemiological tool for studying the spread of P. multocida.
Pasteurella multocida is the cause of disease in a variety of mammalian and avian species. In recent years, toxigenic P. multocida subsp. multocida has been recognized as the cause of progressive atrophic rhinitis in pigs (13). The epidemiology and spread of this organism are, however, not well understood, mainly due to lack of a suitable typing
isolated from 73 pig herds. The strains originated from specific-pathogen-free herds freé of progressive atrophic rhinitis as well as from conventional herds. Capsular types and toxin production are shown in Table 1. Furthermore, 388 strains were examined for phage production. The strains were mainly isolated from pigs, but 1 originated from a cat and 53 were of human origin. Field isolates. Isolates of toxigenic P. multocida were obtained from 37 Danish sow herds. Nasal swabs from 10 4-week-old and 10 8-week-old pigs were spread on selective blood agar plates (1), followed by subcultivation of P. multocida-like colonies. Subcultures were identified and characterized as described below. Identification of strains. P. multocida subsp. multocida strains were identified as described by Foged et al. (7). Toxin production. P. multocida strains were tested for toxin production in a cell culture assay as described by Rutter and Luther (15). Capsular type. Nonserological methods (4, 5) were used to determine capsular type A or D. Media. Subcultivation was performed either on 5% blood agar plates or in tryptic broth. Phage typing was investigated on extract agar plates with CaCl2 (2) or Levinthal agar prepared by adding 100 ml of defibrinated horse blood to 1,200 ml of Difco brain heart infusion agar. After filtration at 80°C, 15 ml of glucose (33%), 10 ml of yeast extract (50%), 4 ml of NAD solution (5%), and 2 ml of CaCl2 2H20 (10%) were added at 45°C. This agar was prepared with or without CaC12. Chemicals. Mitomycin C was obtained from Sigma Chemical Co. and dissolved in saline (10 ,xg/ml). Hyaluronidase (lyophilized from bovine testis) was also from Sigma (batch H-3506; 295 NF units/mg of solid), and a 500-U/ml solution in saline was used. Phage release by mitomycin treatment (reverse typing). Phage induction was performed as described by de Saxe and Notley (6). An overnight broth culture of the strain was diluted 1/100 in fresh tryptic broth and shaken for 2 h at 37°C. To 3.8 ml of the broth culture was added 0.2 ml of mitomycin solution (10 ,ug/ml), and incubation was continued for 30 min. After centrifugation, the bacterial pellet was suspended
system.
Serotyping based on capsular antigen has been used for several years (3, 14), but strains associated with progressive atrophic rhinitis belong to capsular type A or D (7); thus, no sufficient discrimination can be obtained. Also, serotyping based on somatic antigens has been of limited value for epidemiological studies of progressive atrophic rhinitis (14). Investigations on cell envelope proteins and lipopolysaccharides have revealed only a limited number of P. multocida clones (9). The occurrence of bacteriophages in P. multocida has been reported earlier (8). Phage typing has been widely used for typing salmonella, staphylococci, and pseudomonads (12). Phage typing is based on differences in the ability of a number of different phages to lyse, or not lyse, bacterial strains. The purpose of this study was to develop a P. multocida phage typing system which could fulfill the requirements of good discriminatory power, high typability, and good reproducibility. Such a system would be useful in epidemiological studies of the spread of P. multocida both within and between herds and thereby identify sources of infection in pig herds previously free of toxigenic P. multocida. Furthermore, the system might be used as a typing system in infections caused by nontoxigenic P. multocida in other mammalian and avian species.
-
MATERIALS AND METHODS Strains. To develop the phage set, a total of 485 strains of P. multocida subsp. multocida were examined (10). Lytic phages (plaques) were demonstrated on a test set of 97 of these strains of P. multocida, consisting of 3 laboratory strains (NCTC 12177, 4158, and NCTC 12178) and 94 strains *
Corresponding author. 103
J. CLIN. MICROBIOL.
NIELSEN AND ROSDAHL
104
stage, plate cultures could be treated with chloroform vapors for 0.5 h to kill bacterial growth without inactivating the phages. After centrifugation and filtration, 10-fold dilutions of the phage suspension were prepared and 0.02 ml of each dilution was placed on a plate, flooded with the propagating strain. After incubation and determination of PFU per milliliter, we calculated the RTD (i.e., the dilution which contains in 1 drop enough phages for nearly confluent lysis). If the phage suspension was
