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Laboratory Animals (1991) 25, 236-241
Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates RONALD F DIGIACOMO,l
VIVIEN ALLEN2 & MICHAEL H HINTON2
IDepartment of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA 98195, USA, and 2Department of Veterinary Medicine, School of Veterinary Science, University of Bristol, Langford, Avon BS18 7DU, UK
Summary Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.
Materials and methods Animals For several years, the Department of Animal Husbandry School of Veterinary Science, University of Bristol, has had a closed breeding colony of New Zealand White rabbits (Oryctolagus cuniculus). The breeding colony consisted of 45 does and 8 bucks. Litters were weaned at 30 days of age. Subsequently, each litter was reared together as a group. Rabbits were maintained in suspended wire cages in one building. Commercially prepared rabbit feed and water were available ad libitum. Twelve litters of rabbits, aged 35 to 60 days old, comprising 41 rabbits were sampled weekly for 10 to 12 weeks. These litters had no evidence of P. multocida subsp. multocida infection on the initial nasal swab.
Keywords: Pasteurella multocida subsp. multocida; Pasteurellosis, rabbits, typing
Bacterial isolation The nares of rabbits were cultured weekly by inserting a swab into both nasal cavities. The swabs were used to inoculate nutrient agar containing 5OJodefibrinated sheep blood with and without 2 Itg/ml clindamycin (Garlinghouse et al., 1981). Plates were incubated aerobically at 37°C, and examined after 24 and 48 h. Isolates of P. multocida subsp. multocida were identified by colonial morphology, reaction and appearance after gram staining, and reactions with catalase, cytochrome oxidase, indole, urease and growth on bile lactose agar (Cowan, 1974; Mutters et al., 1985).
Several typing systems have been used to characterize Pasteurella multocida subsp. multocida isolates from rabbits including biotyping, antibiograms and capsular and somatic antigen typing (Sato et al., 1967; Lu, Ringler & Park, 1978; Brogden, 1980; Mushin & Schoenbaum, 1980; Hippe & Schliesser, 1981; Percy et al., 1984). However, in most reports, isolates were obtained from diverse sources. In this study, we undertook to determine the variability among P. multocida subsp. multocida isolates from a closed colony of rabbits. Received 2 August 1989; accepted 3 December 1990
Typing of isolates Individual colonies of P. multocida subsp. multocida were picked off the original isolation
Downloaded from lan.sagepub.com at Queen's University on March 7, 2015
Characteristics of P. multocida isolates
plates and streaked onto 5% sheep blood agar and incubated at 37°C for 24 h. Colonies of P. multocida subsp. multocida from each of 5 subcultured plates for each isolate were selected for characterization using the following typing systems: colonial morphology, capsular and somatic antigen typing, biotyping and antimicrobial susceptibility. Two types of colonies were recognized on sheep blood agar: smooth, circular, iridescent colonies and mucoid colonies, which were whitish and irregularly shaped (Carter, 1967; Brogden, 1980). The capsular type of P. multocida subsp. multocida was determined by the staphylococcal hyaluronidase decapsulation (Carter & Rundell, 1975) and acriflavine flocculation (Carter & Subronto, 1973) tests. Somatic type was determined using the gel diffusion precipitin test (Heddleston et al., 1972), by the National Veterinary Services Laboratory, Ames, lA, USA. The biotyping scheme entailed the fermentation reactions (Hinton et al., 1982)to 28 carbohydrates including D( - )arabinose, L( + )arabinose, ,B-D( - )fructose, D( + )galactose, D( + )glucose, a-L-rhamnose, L( - )sorbose, D( + )xylose, L( - )xylose, D( + )cellobiose, 2-deoxy-D-galactose, 2-deoxy-Dglucose, 1-0-methyl-a-D-glucopyranoside, alphalactose, maltose, D( + )mannose, D( + )melibiose, D( + )melizitose, D( + )trehalose dihydrate monohydride, D( + )turanose, sucrose, D( + )raffinose, adonitol, dulcitol, D-mannitol, D-sorbitol, xylitol and myo-inositol together with ornithine decarboxylation and inhibition of growth by 3 dyes, namely, methylene blue (1: 200 000), pyronin (1: 100000) and victor blue (1: 100000). All test media were inoculated from a pure culture master plate with a multi-pronged inoculator (Hinton et al., 1982). The results of carbohydrate fermentation and other tests were recorded after 24 h incubation at 37°C. The antibiotic susceptibility of isolates was determined using the disk diffusion method (Howe & Linton, 1976). The media used was isosensitest agar (Oxoid Ltd, Basingstoke, UK) supplemented with 2070(v/v) fetal calf serum. The antibacterial sensitivity disks (Oxoid Ltd, Basingstoke, UK) used included ampicillin (25 j.tg), chloramphenicol (50 j.tg),
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colistin sulphate (10 j.tg), kanamycin (30 j.tg) nalidixic acid (30 j.tg), nitrofurantoin (200 j.tg), streptomycin (25 j.tg),sulphafurazole (500 j.tg)and tetracycline (50 j.tg). The diameters of zones of inhibition of bacterial growth around antibiotic disks were measured after 24 h. Isolates with inhibition zones < 2 mm from the 6 mm disk were considered resistant. Results Eleven of 19 (58070)rabbits in 5 of the 12 litters acquired P. multocida subsp. multocida infection. Isolates with smooth iridescent colonies were recovered from infected rabbits in 3 litters whereas mucoid colonies were isolated from infected rabbits in 2 litters (Table 1). The smooth iridescent colonies from the 3 litters were nontypeable, negative in both the hyaluronidase inhibition and acriflavine flocculation tests, whereas the mucoid colonies from the 2 litters were capsular type A (Table 1). Two basic patterns of carbohydrate fermentation were demonstrated (Table 2). Isolates from rabbits 898-1 and 903-2 fermented fructose, galactose, glucose, xylose, maltose, mannose, manitol, sorbitol and sucrose. Most isolates also fermented turanose and reacted with victor blue. A similar pattern of fermentation reactions was observed with isolates from rabbit 897-1, except that they also decarboxylated ornithine. The pattern of two littermates, 897-2 and 897-4 differed slightly, particularly with later isolates, since they also fermented arabinose, sorbose and xylitol but did not react with victor blue. Isolates Table 1. Cultural characteristics and capsular typing of Pasteurella multocida subsp. multocida isolates from rabbits Liller
No. of rabbits"
898 903 897 899
1/1 1/2 3/5 4/5
894
2/6
No. of isolateS'
Colonial morphology
Capsular type A D
5
51 51 51 M M
Non-typeable Non-typeable Non-typeable
6 5, 6, 8 3,3,4,4 2,2
+ +
'Number infected/number in litter. bNumber of P. multocida subsp. multocida isolates obtained from each rabbit. Five colonies of each isolate were selected for testing. 'SI = smooth iridescent, M = mucoid.
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Laboratory Animals (1991) 25, 236-241
Naturally acquired Pasteurella multocida subsp. multocida infection in a closed colony of rabbits: characteristics of isolates RONALD F DIGIACOMO,l
VIVIEN ALLEN2 & MICHAEL H HINTON2
IDepartment of Comparative Medicine, School of Medicine, University of Washington, Seattle, WA 98195, USA, and 2Department of Veterinary Medicine, School of Veterinary Science, University of Bristol, Langford, Avon BS18 7DU, UK
Summary Twelve litters, comprising 41 rabbits aged 35 to 60 days old, in a closed university colony, were monitored for acquisition of nasal Pasteurella multocida subsp. multocida infection. Isolates from 11 infected rabbits were characterized by colonial morphology, capsular type, biotype and antibiotic resistance. Selected isolates were further characterized by somatic antigen typing. Two major strains of P. multocida subsp. multocida were detected in the colony. One strain had mucoid colonies, fermented few carbohydrates and was serotype A:5, whereas, the other strain had smooth iridescent colonies, non-typeable capsular antigen, type 3 somatic antigen and fermented more than twice as many carbohydrates.
Materials and methods Animals For several years, the Department of Animal Husbandry School of Veterinary Science, University of Bristol, has had a closed breeding colony of New Zealand White rabbits (Oryctolagus cuniculus). The breeding colony consisted of 45 does and 8 bucks. Litters were weaned at 30 days of age. Subsequently, each litter was reared together as a group. Rabbits were maintained in suspended wire cages in one building. Commercially prepared rabbit feed and water were available ad libitum. Twelve litters of rabbits, aged 35 to 60 days old, comprising 41 rabbits were sampled weekly for 10 to 12 weeks. These litters had no evidence of P. multocida subsp. multocida infection on the initial nasal swab.
Keywords: Pasteurella multocida subsp. multocida; Pasteurellosis, rabbits, typing
Bacterial isolation The nares of rabbits were cultured weekly by inserting a swab into both nasal cavities. The swabs were used to inoculate nutrient agar containing 5OJodefibrinated sheep blood with and without 2 Itg/ml clindamycin (Garlinghouse et al., 1981). Plates were incubated aerobically at 37°C, and examined after 24 and 48 h. Isolates of P. multocida subsp. multocida were identified by colonial morphology, reaction and appearance after gram staining, and reactions with catalase, cytochrome oxidase, indole, urease and growth on bile lactose agar (Cowan, 1974; Mutters et al., 1985).
Several typing systems have been used to characterize Pasteurella multocida subsp. multocida isolates from rabbits including biotyping, antibiograms and capsular and somatic antigen typing (Sato et al., 1967; Lu, Ringler & Park, 1978; Brogden, 1980; Mushin & Schoenbaum, 1980; Hippe & Schliesser, 1981; Percy et al., 1984). However, in most reports, isolates were obtained from diverse sources. In this study, we undertook to determine the variability among P. multocida subsp. multocida isolates from a closed colony of rabbits. Received 2 August 1989; accepted 3 December 1990
Typing of isolates Individual colonies of P. multocida subsp. multocida were picked off the original isolation
Downloaded from lan.sagepub.com at Queen's University on March 7, 2015
Characteristics of P. multocida isolates
plates and streaked onto 5% sheep blood agar and incubated at 37°C for 24 h. Colonies of P. multocida subsp. multocida from each of 5 subcultured plates for each isolate were selected for characterization using the following typing systems: colonial morphology, capsular and somatic antigen typing, biotyping and antimicrobial susceptibility. Two types of colonies were recognized on sheep blood agar: smooth, circular, iridescent colonies and mucoid colonies, which were whitish and irregularly shaped (Carter, 1967; Brogden, 1980). The capsular type of P. multocida subsp. multocida was determined by the staphylococcal hyaluronidase decapsulation (Carter & Rundell, 1975) and acriflavine flocculation (Carter & Subronto, 1973) tests. Somatic type was determined using the gel diffusion precipitin test (Heddleston et al., 1972), by the National Veterinary Services Laboratory, Ames, lA, USA. The biotyping scheme entailed the fermentation reactions (Hinton et al., 1982)to 28 carbohydrates including D( - )arabinose, L( + )arabinose, ,B-D( - )fructose, D( + )galactose, D( + )glucose, a-L-rhamnose, L( - )sorbose, D( + )xylose, L( - )xylose, D( + )cellobiose, 2-deoxy-D-galactose, 2-deoxy-Dglucose, 1-0-methyl-a-D-glucopyranoside, alphalactose, maltose, D( + )mannose, D( + )melibiose, D( + )melizitose, D( + )trehalose dihydrate monohydride, D( + )turanose, sucrose, D( + )raffinose, adonitol, dulcitol, D-mannitol, D-sorbitol, xylitol and myo-inositol together with ornithine decarboxylation and inhibition of growth by 3 dyes, namely, methylene blue (1: 200 000), pyronin (1: 100000) and victor blue (1: 100000). All test media were inoculated from a pure culture master plate with a multi-pronged inoculator (Hinton et al., 1982). The results of carbohydrate fermentation and other tests were recorded after 24 h incubation at 37°C. The antibiotic susceptibility of isolates was determined using the disk diffusion method (Howe & Linton, 1976). The media used was isosensitest agar (Oxoid Ltd, Basingstoke, UK) supplemented with 2070(v/v) fetal calf serum. The antibacterial sensitivity disks (Oxoid Ltd, Basingstoke, UK) used included ampicillin (25 j.tg), chloramphenicol (50 j.tg),
237
colistin sulphate (10 j.tg), kanamycin (30 j.tg) nalidixic acid (30 j.tg), nitrofurantoin (200 j.tg), streptomycin (25 j.tg),sulphafurazole (500 j.tg)and tetracycline (50 j.tg). The diameters of zones of inhibition of bacterial growth around antibiotic disks were measured after 24 h. Isolates with inhibition zones < 2 mm from the 6 mm disk were considered resistant. Results Eleven of 19 (58070)rabbits in 5 of the 12 litters acquired P. multocida subsp. multocida infection. Isolates with smooth iridescent colonies were recovered from infected rabbits in 3 litters whereas mucoid colonies were isolated from infected rabbits in 2 litters (Table 1). The smooth iridescent colonies from the 3 litters were nontypeable, negative in both the hyaluronidase inhibition and acriflavine flocculation tests, whereas the mucoid colonies from the 2 litters were capsular type A (Table 1). Two basic patterns of carbohydrate fermentation were demonstrated (Table 2). Isolates from rabbits 898-1 and 903-2 fermented fructose, galactose, glucose, xylose, maltose, mannose, manitol, sorbitol and sucrose. Most isolates also fermented turanose and reacted with victor blue. A similar pattern of fermentation reactions was observed with isolates from rabbit 897-1, except that they also decarboxylated ornithine. The pattern of two littermates, 897-2 and 897-4 differed slightly, particularly with later isolates, since they also fermented arabinose, sorbose and xylitol but did not react with victor blue. Isolates Table 1. Cultural characteristics and capsular typing of Pasteurella multocida subsp. multocida isolates from rabbits Liller
No. of rabbits"
898 903 897 899
1/1 1/2 3/5 4/5
894
2/6
No. of isolateS'
Colonial morphology
Capsular type A D
5
51 51 51 M M
Non-typeable Non-typeable Non-typeable
6 5, 6, 8 3,3,4,4 2,2
+ +
'Number infected/number in litter. bNumber of P. multocida subsp. multocida isolates obtained from each rabbit. Five colonies of each isolate were selected for testing. 'SI = smooth iridescent, M = mucoid.
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DiGiacomo, Allen & Hinton
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