Modification of Cary-Blair Transport Medium for Pasteurella multocida and Mannheimia haemolytica

Tefera G. , J . Smola: Modification of Cary-Blair Transport Medium for Pasteurella multocida and Mannheimia haemolytica. Acta Vet. Brno 2002, 71: 229–233. The aim of this study was to evaluate the effectiveness of Cary-Blair, Amies and modified Cary-Blair medium for transport of P. multocida and M. haemolytica strains. Pure cultures of nine strains of P. multocida, representing all subspecies, and nine strains of M. haemolytica were inoculated into the media to test their convenience. The ability of the strains to survive in each of these media was determined periodically (on days 5, 10, 15, 20, 30, 45, 60, 75, 90, respectively) at different temperatures (alternatively at 21 °C and 4 °C) and at room temperature (constantly at 21 °C). Comparing with the Cary-Blair and Amies medium, P. multocida strains survived for 45 days and M. haemolytica strains for 30 days in modified Cary-Blair medium. Based on the present observation, Cary-Blair medium modified by the addition of horse blood serum is the most effective of those tested media for the transport of P. multocida and M. haemolytica strains. Transport medium, P. multocida, M. haemolytica A transport medium convenient for Pasteurella and Mannheimia has not been well investigated and established. For the first time Percy et al. (1984) used Cary-Blair medium as the transport medium for a survey of serotypes of P. multocida, and suggested the use of this medium to transport respiratory pathogens. Furthermore, Sh imoda et al. (1991) analyzed the stability of pathogenic bacteria from laboratory animals in various transport media and showed that with phosphate-buffered saline (PBS) and PBS it was difficult to maintain Pasteurella strains at various temperatures (37 °C, 24 °C and 4 °C). Recently, by comparing eight transport media, Kawamoto et al. (1997) showed that Cary-Blair medium and Leibovitz medium No. 15 are the effective media to transport P. multocida strains. During this investigation, it was indicated that Cary-Blair medium is superior for the transport of strains of Pasteurella spp. isolated from animals and humans. Based on this finding, the present study was carried out to evaluate Cary-Blair, Amies and modified Cary-Blair medium for safe transport of P. multocida and M. haemolytica Materials and Methods Bacter ia l s t ra ins A total of eighteen strains of P. multocida and M. haemolytica were used in this study. Two of them were type strains and eight of them were reference strains. Both type and reference strains were obtained from CCM (Czech Collection of Microorganisms). The remaining eight strains, which were identified using RapiD 20 E (bioMérieux, France), were isolated from fatal infectious cases of cattle, pigs and poultry (Table 1). Swabs Synthetic-tipped individually wrapped cotton swabs sterilized by ionizing radiation (Copanitalia, Italy) were used to collect field samples and pure cultures of P. multocida and M. haemolytica. ACTA VET. BRNO 2002, 71: 229–233 Address for correspondence: Dr. Genene Tefera Department of Microbiology and Immunology University of Veterinary and Pharmaceutical Sciences Palackého 1/3 Brno, Czech Republic Phone: +420 5 4156 2282 Fax: +420 5 4121 2646 Email: teferag@vfu.cz http:/www.vfu.cz/acta-vet/actavet.htm Bacter iological Media Transport media Cary-Blair transport medium (CM519, Oxoid) and Amies transport medium (CM425, Oxoid) The media were prepared according to the manufacturer’s instructions (Oxoid) by distributing 1.5 ml into Nunc Cryotubes (Denmark) and sterilized by immersing in free-steam for 15 minutes. The tubes were allowed to cool and tightened to prevent water loss. Modif ied Cary-Blair medium The medium was prepared by adding 5% v/v of sterile horse blood serum to Cary-Blair medium (Oxoid) at 50 °C and distributing 1.5 ml into tubes. The tubes were cooled and tightened to prevent water loss. Cul ture medium Sheep blood agar was prepared according to the manufacturer by adding sheep blood (5% v/v) to the Columbia blood agar base (CM351, Oxoid). Inoculat ion, s torage and survival checking All type, reference and field strains of P. multocida and M. haemolytica were routinely cultured on sheep blood agar at 37 °C. Four colonies of P. multocida and M. haemolytica, taken from fresh overnight cultures of each strain, were transferred using sterile loops to Cary-Blair medium, modified Cary-Blair medium and Amies transport medium. These media were stored at different temperatures (alternatively at 21 °C and 4 °C) and at room temperature (constantly at 21 °C) for a period of 45-90 days. The strains were periodically recultivated on sheep blood agar using sterile loops by forming 5 overlapping segments to check their survival ability based on the quantity of bacterial cells. The plates were incubated aerobically at 37 °C for 24 h. Reading of resul ts Survival ability of P. multocida and M. haemolytica strains was checked at periodical intervals of days (i.e. on day 5, 10, 15, 20, 30, 45 (experiment 1, 2, 3), 60 (Experiment 2, 3), 75 and 90 (Experiment 4)). The evaluation was conducted visually according to the growth of each strain on sheep blood agar plates (Claus 1981). The presence of growth of each strain on streaked segments of blood agar was classified as positive and its absence as negative. Design of experiment The ability of P. multocida and M. haemolytica strains to survive on the tested transport media was observed in four experimental stages. All eighteen strains were inoculated in Cary-Blair medium and their survival ability was observed for 45 days at different temperatures (alternatively at 21 °C and 4 °C) (Experiment 1). These media were stored alternatively at 21 °C and 4 °C every day in order to simulate with the conditions in which the specimens are collected and transported. On the other hand, the same strains of P. multocida and M. haemolytica 230 Table 1 Type, reference and field strains of P. multocida and M. haemolytica used for this study Species Origin Strains P. multocida Bovine type (CCM 5903) P. multocida Bovine reference (CCM 6081) P. multocida ssp. septica Bovine reference (CCM 4730) P. multocida ssp. gallicida Bovine reference (CCM 4731) P. multocida ssp. multocida Bovine reference (CCM 5419) P. multocida Porcine field (8891) P. multocida Porcine field (8967) P. multocida Avian field (9506) P. multocida Bovine field (10338) M. haemolytica Bovine type (CCM 5141) M. haemolytica Bovine reference (CCM 6169) M. haemolytica Ovine reference (CCM 6170) M. haemolytica Ovine reference (CCM 6174) M. haemolytica Ovine reference (CCM 6176) M. haemolytica Bovine field (9122) M. haemolytica Bovine field (10339) M. haemolytica Bovine field (10517) M. haemolytica Bovine field (10518) were observed for 60 days in Cary-Blair (Experiment 2) and Amies medium (Experiment 3) at room temperature (constantly at 21 °C), and they were observed for 90 days in the modified Cary-Blair medium at room temperature (Experiment 4).


Bacteriological Media Transport media
Cary-Blair transport medium (CM519, Oxoid) and Amies transport medium (CM425, Oxoid) The media were prepared according to the manufacturer's instructions (Oxoid) by distributing 1.5 ml into Nunc Cryotubes (Denmark) and sterilized by immersing in free-steam for 15 minutes.The tubes were allowed to cool and tightened to prevent water loss.

Modified Cary-Blair medium
The medium was prepared by adding 5% v/v of sterile horse blood serum to Cary-Blair medium (Oxoid) at 50 °C and distributing 1.5 ml into tubes.The tubes were cooled and tightened to prevent water loss.

Culture medium
Sheep blood agar was prepared according to the manufacturer by adding sheep blood (5% v/v) to the Columbia blood agar base (CM351, Oxoid).

Inoculation, storage and survival checking
All type, reference and field strains of P. multocida and M. haemolytica were routinely cultured on sheep blood agar at 37 °C.Four colonies of P. multocida and M. haemolytica, taken from fresh overnight cultures of each strain, were transferred using sterile loops to Cary-Blair medium, modified Cary-Blair medium and Amies transport medium.These media were stored at different temperatures (alternatively at 21 °C and 4 °C) and at room temperature (constantly at 21 °C) for a period of 45-90 days.The strains were periodically recultivated on sheep blood agar using sterile loops by forming 5 overlapping segments to check their survival ability based on the quantity of bacterial cells.The plates were incubated aerobically at 37 °C for 24 h.

Reading of results
Survival ability of P. multocida and M. haemolytica strains was checked at periodical intervals of days (i.e. on day 5, 10, 15, 20, 30, 45 (experiment 1, 2, 3), 60 (Experiment 2, 3), 75 and 90 (Experiment 4)).The evaluation was conducted visually according to the growth of each strain on sheep blood agar plates (Claus 1981).The presence of growth of each strain on streaked segments of blood agar was classified as positive and its absence as negative.

Design of experiment
The ability of P. multocida and M. haemolytica strains to survive on the tested transport media was observed in four experimental stages.All eighteen strains were inoculated in Cary-Blair medium and their survival ability was observed for 45 days at different temperatures (alternatively at 21 °C and 4 °C) (Experiment 1).These media were stored alternatively at 21 °C and 4 °C every day in order to simulate with the conditions in which the specimens are collected and transported.On the other hand, the same strains of P. multocida and M. haemolytica  4) + ( 4)  4) + ( 4) + ( 4) + ( 4) + (4) --field 4 + (4) + ( 4) + ( 4) + ( 4) + ( 4) + ( 4) + ( 4) + ( 4) - M. haemolytica reference 4 + (4) + ( 4) + ( 4) + ( 4) + ( 4) ---field 4 + (4) + ( 4) + ( 4) + ( 4) + ( 4) + (4) --- In conclusion: compared with Cary-Blair and Amies transport medium, all P. multocida strains survived for 45 days and all M. haemolytica strains for 30 days in the modified Cary-Blair medium.The results of this study showed that modified Cary-Blair medium is superior to the other media tested with regard to survival of P. multocida and M. haemolytica in pure cultures and field specimens isolated in the Czech Republic and Ethiopia.Based on these findings, we recommend the use of the modified Cary-Blair transport medium for P. multocida and M. haemolytica strains, as well as its use for other bacterial species of the family Pasteurellaceae.

Table 2
The ability of P. multocida and M. haemolytica strains to survive in Cary-Blair medium at different temperatures (alternatively at 21 °C and 4 °C)

Table 4
The ability of P. multocida and M. haemolytica strains to survive in modified Cary-Blair medium at room temperature(21 °C)