Gavin Zealey

Electrotransformation of Bordetella

Whooping cough (pertussis) is a disease of the upper respiratory tract caused by a localized infection by B. pertussis. The absence of a technique for high-frequency transformation has limited the genetic manipulation of this organism. Weiss and Falkow (1982) described that B. pertussis cells cannot be transformed by classical methods such as calcium chloride treatment but that a cold shock or freezing of the cells results in transformation by plasmids of the P and W incompatibility groups. The transformation frequency obtained was less than 103/µg of DNA however and a restriction system in B. pertussis prevents the introduction of plasmids containing the Hind III recognition sequence. A modified form of this technique has been used to transform B. bronchiseptica at a frequency of 104/µg of DNA (Lax, 1987). Exposure of living cells to brief pulses of electric current results in a reversible permeabilisation of the biomembranes and has been termed electroporation. This transient membrane permeability then allows DNA uptake by the cells. We describe here the applicability of electroporation to the transformation of Bordetella pertussis and B. parapertussis. Electrotransformation routinely yields > 106 transformants per µg of plasmid DNA.

The New Generation of Recombinant Pertussis Vaccines

Whooping cough or pertussis is a severe, highly contagious upper respiratory tract infections, mainly affecting young children. The World Health Organization estimates that there are 60 million cases of pertussis per year and 0.5-1 million associated deaths (Muller et al., 1986). In unvaccinated populations, an incidence rate as high as 80% has been observed in children under 5 years old (Fine and Clarkson, 1984). The disease has two distinct phases, the early phase or catarrhal stage, lasts up to two weeks with symptoms of low-grade fever and mild cough. The second phase is the paroxysmal stage which can last for 4–6 weeks and is accompanied by the characteristic whoop. Infants, in particular, an suffer apnoea and hypoxaemia during these prolonged coughing bouts and there is a 5% incidence of serious neurological complications. Although pertussis is considered to be a childhood disease, there is increasing evidence of clinical and asymptomatic disease in adolescents and adults (Mortimer, 1990; Addiss et al., 1991; Halperin and Marrie, 1991). Erythromycin treatment is effective in clearing bacteria but has little effect on symptoms. Passive immunotherapy, on the other hand has been shown to reduce the frequency and severity of whoops (Granstrom et al., 1991).