The mRNA expression of several cytokines was evaluated in splenocytes and mesenteric lymph node (MLN) cells of rats infected with Capillaria hepatica by reverse-transcription (RT)-PCR until week 12 after infection. IgG1 and IgG2a, which are associated with Th1 and Th2 response, respectively, were also assessed by ELISA. The results indicated that the majority of cytokines, including the Th1 (IL-2 and IFN-γ) and Th2 cytokines (IL-4, IL-5 and IL-10) were expressed at maximal levels during the early stage of infection (after week 1-2), and the ELISA data also evidenced a similar pattern of changes in IgG1 and IgG2a. Th1 and Th2 cytokines responded in a similar fashion in this rat model. The expression of cytokines in splenocytes was significantly higher than that in MLN cells, thereby indicating that cytokine production is controlled more by spleen than by MLN. In addition, the observation that IFN-γ expression increased unexpectedly at the time of maximal egg production (6 weeks after infection) indicated that IFN-γ is a cytokine reacting against egg production. However, increased IL-5 expression occurring in tandem with worm activity indicated that the activity of C. hepatica might be controlled by IL-5 expression.

Changes in the expression level of splenocyte IFN-γ mRNA of Sprague-Dawley (SD) rats infected with Paragonimus westermani were analyzed by competitive reverse transcription-polymerase chain reaction (RT-PCR) followed by southern blot. The template RNA was extracted from the splenocytes of rats infected with 20 metacercariae of P. westermani. The products of competitive RT-PCR were subjected to southern blot and enhanced chemiluminescence (ECL), and analyzed with a densitometer. In comparison with that of uninfected control rat splenocytes (value of 1), the levels of mRNA expression of IFN-γ had changed to 0.747 at 1 week post infection (PI), 0.00175 at 2 week PI, 0.0217 at 3 week PI, 0.194 at 4 week PI and then to 0.537 at 5 week PI. The level at 7 week PI had returned to 1.25, comparable with that of uninfected rats. These results show that, when infected with P. westermani, the levels of IFN-γ mRNA of SD rat splenocytes were remarkably reduced by more than 500 times at 2 week PI and restored to normal level at 7 week PI.

Identification of the genes responsible for the recovery of virulence in brain-passaged Acanthamoeba culbertsoni was attempted via mRNA differential display-polymerase chain reaction (mRNA DD-PCR) analysis. In order to identify the regulatory changes in transcription of the virulence related genes by the brain passages, mRNA DD-PCR was performed which enabled the display of differentially transcribed mRNAs after the brain passages. Through mRNA DD-PCR analysis, 96 brain-passaged amoeba specific amplicons were observed and were screened to identify the amplicons that failed to amplify in the non-brain-passaged amoeba mRNAs. Out of the 96 brain-passaged amoeba specific amplicons, 12 turned out to be amplified only from the brain-passaged amoeba mRNAs by DNA slot blot hybridization. The clone, A289C, amplified with an arbitrary primer of UBC #289 and the oligo dT₁₁-C primer, revealed the highest homology (49.8%) to the amino acid sequences of UPD-galactose lipid transferase of Erwinia amylovora, which is known to act as an important virulence factor. The deduced amino acid sequences of an insert DNA in clone A289C were also revealed to be similar to cpsD, which is the essential gene for the expression of type III capsule in group B streptococcus. Upregulated expression of clone A289C was verified by RNA slot blot hybridization. Similar hydrophobicity values were also observed between A289C (at residues 47-66) and the AmsG gene of E. amylovora (at residues 286-305: transmembrane domains). This result suggested that the insert of clone A289C might play the same function as galactosyl transferase controlled by the AmsG gene in E. amylovora.