The role of cofeeding arthropods in the transmission of Rickettsia felis

Rickettsia felis is an emerging etiological agent of rickettsioses worldwide. The cosmopolitan cat flea (Ctenocephalides felis) is the primary vector of R. felis, but R. felis has also been reported in other species of hematophagous arthropods including ticks and mosquitoes. Canines can serve as a bacteremic host to infect fleas under laboratory conditions, yet isolation of R. felis from the blood of a vertebrate host in nature has not been realized. Cofeeding transmission is an efficient mechanism for transmitting rickettsiae between infected and uninfected fleas; however, the mechanism of transmission among different orders and classes of arthropods is not known. The potential for R. felis transmission between infected fleas and tick (Dermacentor variabilis) and mosquito (Anopheles quadrimaculatus) hosts was examined via cofeeding bioassays. Donor cat fleas infected with R. felis transmitted the agent to naïve D. variabilis nymphs via cofeeding on a rat host. Subsequent transstadial transmission of R. felis from the engorged nymphs to the adult ticks was observed with reduced prevalence in adult ticks. Using an artificial host system, An. quadrimaculatus exposed to a R. felis-infected blood meal acquired rickettsiae and maintained infection over 12 days post-exposure (dpe). Similar to ticks, mosquitoes were able to acquire R. felis while cofeeding with infected cat fleas on rats infection persisting in the mosquito for up to 3 dpe. The results indicate R. felis-infected cat fleas can transmit rickettsiae to both ticks and mosquitoes via cofeeding on a vertebrate host, thus providing a potential avenue for the diversity of R. felis-infected arthropods in nature.     

Expression of human pyruvate carboxylase in insect cells using the baculovirus system

Constructs containing cDNA encoding human pyruvate carboxylase (PC) with and without a hexahistidine (6x His) tag at the N-terminal of the mature enzyme have been cloned under the control of the polyhedrin promoter. These two constructs were co-transfected with the baculovirus genome into Sf9 cells to produce recombinant baculoviruses harbouring human PC cDNA. The expression of human PC under the control of the polyhedrin promoter was found to be at its highest level at 4 days post-infection. The expressed material accounted for up to 70% of total cellular protein with 5% of this expressed material being found in the soluble fraction. The recombinant human 6x His-PC isolated with a purity of approximately 50% using a Ni-NTA agarose column was found to have the specific activity of 7U/mg, which was similar to that produced from a 293T stable line [Biochem. Biophys. Res. Commun. 266 (1999) 512]. This is the first report of a heterologous expression system for recombinant human PC.