Detection of Plasmodium yoelii stage mRNA in BALB/c mice

Relatively little information is available concerning th expression of para site genes during the liver stage of Plasmodium infection, mostly because o f low-level infection of host hepatocytes and the lack of purification tech niques for the liver stage parasites. We have determined the optimal dosage of Plasmodium yoelii sporozoite inoculum and routes of inoculation, which are intravenous tail vein and the intrahepatic portal circulation. To deter mine which route was optimal, BALB/c mice were inoculated via 1 of these ro utes, and parasitemia was detected by reverse transcription polymerase chai n reaction (RT-PCR) detecting both murine beta -actin and P. yoelii-specifi c 28S ribosomal RNA in the liver samples. Murine beta -actin was detected a fter 15 cycles of PCR, and its expression levels did not differ between tre atment groups. However, P. yoelii-specific 28S ribosomal RNA gene product w as detected after 15 cycles of PCR in animals inoculated via the tail vein but was not detected until 25 cycles in animals inoculated via the intrahep atic portal circulation. Experiments were then performed to identify the sm allest inoculum required to initiate a liver stage infection that would yie ld sufficient parasite RNA for analysis. Inoculation with different doses o f sporozoite inocula was followed by RT-PCR on the livers of the inoculated animals. The P. yoelii-specific 28S ribosomal RNA gene product was first d etected in both treatment groups after 15 cycles, suggesting that both dose s of sporozoite inocula provided relatively the same level of liver infecti on rate. We also have analyzed infected mouse liver for parasite-specific m RNA, which was detectable as early as 24 hr postinfection.