Laboratory Animals (1976) 10,47-48.
47
PROBLEMS ASSOCIATED WITH THE INDENTIFICATION BORDETELLA
OF
BRONCHISEPTICA by
W. SIMPSON
and D. J. C. SIMMONS
Institute of Cancer Research, Royal Cancer Hospital, Pollards Wood Research Station, Nightingales Lane, Chalfont St Giles, HP8 4SP SUMMARY Bordefella bronchisepfica, isolated from rodent nasopharyngeal swabs, failed to produce characteristic colonies after 24 hours incubation at 37°C. 4-7 days incubation at 37°C was required to achieve positive motility test results, when isolates later identified as B. bronchisepfica were tested by Craigie tube and soft agar stab methods. The biochemical tests used to identify suspected B. bronchisepfica are specified.
When grown on blood agar Bordetella bronchiseptica colonies are usually described as rapidly growing, haemolytic, glistening, 2·0 mm diameter. In our experience this description of colonial morphology is not typical when primary cultures are being examined. Ready poured blood-agar plates (Oxoid Ltd, 20 Southwark Bridge Road, London, SEI 9HF) are used in this laboratory. On this medium B. bronchiseptica isolated on primary culture from rodent nasa-pharyngeal swabs produced tiny nonhaemolytic colonies 0,1-0,2 mm after 24 h incubation at 37°C, increasing to 1·5-2·0 mm after 48 h incubation. The organism was invariably non haemolytic on direct isolation and subculture, which agrees with Pickett & Manclark (1970). The appearance of some isolates of B. bronchiseptica was similar to Neisseria catarrhalis. The smoothness of the Bordetella colonies was however readily distinguished from the roughness of the Neisseria colonies by touching with a loop. These different organisms frequently appear together in cultures from rabbit and guinea-pig nasopharynx. B. bronchiseptica can appear non-motile when tested using Craigie tubes or soft agar stabs. In Craigie tubes the inoculated colony grows only at the top of the centre tube for 1-3 days. Growth then extends slowly down this tube adhering to the glass, and up the outside. When the growth approaches the surface of the medium the organism spreads rapidly through the upper
Downloaded from lan.sagepub.com by guest on March 16, 2015
48
W. SIMPSON
AND
D. J. C. SIMMONS
layers of the medium. This whole process can take up to 7 days. In soft agar stabs the organism extends slowly out from the stab, again taking up to 7 days to show a positive result. The pattern of growth on blood agar and possible misinterpretation of motility tests can lead to confusion with biochemically similar organisms such as Bordetella parapertussis. Tests used to identify suspected B. bronchiseptica isolates and reactions of B. bronchiseptica in this laboratory are given in Table I. Table 1. Primary tests used in the identification of Bordetclla
bronclliseptica.
Test
B. bronchiseptica
Ureaset Oxidase Glucose (acid) O-F Growth on MacConkey agar
+ (almost instantaneous) + (strong)
reaction*
- (alkaline in oxidative tube)
+
*+positive result; -negative result. tUrea diagnostic disks (Difco Laboratories, P.O.B. 148, Central Avenue, East Molesey, Surrey). The primary identification of B. bronchiseptica depends on the almost instantaneous reactions with the urea diagnostic disks, the alkalinisation of glucose in the oxidative tube in the O-F test, the strong oxidase reaction and growth on MacConkey agar. Confirmatory tests used are motility and the alkalinisation of certain amides as described by Pickett & Pedersen (I970a, b). When specimens are specifically examined for the presence of B. bronchiseptica a selective medium may be used (Woode & McLeod, 1967). This is not practicable when large numbers of specimens are being examined for a wide range of organisms.
Pickett, M. J.
&
REFERENCES Manclark, C. R. (1970). Nonfermentative bacilli associated with man.
American Journal of Clinical Pathology 54, 155-163.
Pickett, M. J. & ,Pedersen,M. M. (1970a). Characterization of saccharolytic nonfermentative bacteria associated with man. Canadian Journal of Microbiology 16, 351-362. Pickett, M. J. & Pedersen, M. M. (I 970b). Nonfermentative bacilli associated with man. American Journal of Clinical Pathology 54, 164-177.
Woode, G. N. & McLeod, N. (1967). Control of acute Bordetella bronchiseptica pneumonia in a guinea-pig colony, Laboratory Animals 1, 9.1-94.
Downloaded from lan.sagepub.com by guest on March 16, 2015
47
PROBLEMS ASSOCIATED WITH THE INDENTIFICATION BORDETELLA
OF
BRONCHISEPTICA by
W. SIMPSON
and D. J. C. SIMMONS
Institute of Cancer Research, Royal Cancer Hospital, Pollards Wood Research Station, Nightingales Lane, Chalfont St Giles, HP8 4SP SUMMARY Bordefella bronchisepfica, isolated from rodent nasopharyngeal swabs, failed to produce characteristic colonies after 24 hours incubation at 37°C. 4-7 days incubation at 37°C was required to achieve positive motility test results, when isolates later identified as B. bronchisepfica were tested by Craigie tube and soft agar stab methods. The biochemical tests used to identify suspected B. bronchisepfica are specified.
When grown on blood agar Bordetella bronchiseptica colonies are usually described as rapidly growing, haemolytic, glistening, 2·0 mm diameter. In our experience this description of colonial morphology is not typical when primary cultures are being examined. Ready poured blood-agar plates (Oxoid Ltd, 20 Southwark Bridge Road, London, SEI 9HF) are used in this laboratory. On this medium B. bronchiseptica isolated on primary culture from rodent nasa-pharyngeal swabs produced tiny nonhaemolytic colonies 0,1-0,2 mm after 24 h incubation at 37°C, increasing to 1·5-2·0 mm after 48 h incubation. The organism was invariably non haemolytic on direct isolation and subculture, which agrees with Pickett & Manclark (1970). The appearance of some isolates of B. bronchiseptica was similar to Neisseria catarrhalis. The smoothness of the Bordetella colonies was however readily distinguished from the roughness of the Neisseria colonies by touching with a loop. These different organisms frequently appear together in cultures from rabbit and guinea-pig nasopharynx. B. bronchiseptica can appear non-motile when tested using Craigie tubes or soft agar stabs. In Craigie tubes the inoculated colony grows only at the top of the centre tube for 1-3 days. Growth then extends slowly down this tube adhering to the glass, and up the outside. When the growth approaches the surface of the medium the organism spreads rapidly through the upper
Downloaded from lan.sagepub.com by guest on March 16, 2015
48
W. SIMPSON
AND
D. J. C. SIMMONS
layers of the medium. This whole process can take up to 7 days. In soft agar stabs the organism extends slowly out from the stab, again taking up to 7 days to show a positive result. The pattern of growth on blood agar and possible misinterpretation of motility tests can lead to confusion with biochemically similar organisms such as Bordetella parapertussis. Tests used to identify suspected B. bronchiseptica isolates and reactions of B. bronchiseptica in this laboratory are given in Table I. Table 1. Primary tests used in the identification of Bordetclla
bronclliseptica.
Test
B. bronchiseptica
Ureaset Oxidase Glucose (acid) O-F Growth on MacConkey agar
+ (almost instantaneous) + (strong)
reaction*
- (alkaline in oxidative tube)
+
*+positive result; -negative result. tUrea diagnostic disks (Difco Laboratories, P.O.B. 148, Central Avenue, East Molesey, Surrey). The primary identification of B. bronchiseptica depends on the almost instantaneous reactions with the urea diagnostic disks, the alkalinisation of glucose in the oxidative tube in the O-F test, the strong oxidase reaction and growth on MacConkey agar. Confirmatory tests used are motility and the alkalinisation of certain amides as described by Pickett & Pedersen (I970a, b). When specimens are specifically examined for the presence of B. bronchiseptica a selective medium may be used (Woode & McLeod, 1967). This is not practicable when large numbers of specimens are being examined for a wide range of organisms.
Pickett, M. J.
&
REFERENCES Manclark, C. R. (1970). Nonfermentative bacilli associated with man.
American Journal of Clinical Pathology 54, 155-163.
Pickett, M. J. & ,Pedersen,M. M. (1970a). Characterization of saccharolytic nonfermentative bacteria associated with man. Canadian Journal of Microbiology 16, 351-362. Pickett, M. J. & Pedersen, M. M. (I 970b). Nonfermentative bacilli associated with man. American Journal of Clinical Pathology 54, 164-177.
Woode, G. N. & McLeod, N. (1967). Control of acute Bordetella bronchiseptica pneumonia in a guinea-pig colony, Laboratory Animals 1, 9.1-94.
Downloaded from lan.sagepub.com by guest on March 16, 2015
