Studies On Treatment Of Murine Visceral Leishmaniasis, Cloning And Expression Of LACK Gene From L.d. And Adherence-conferring Factor Of E.coli

Visceral leishmaniasis (VL), is a zoonotic disease caused by protozoa of the Leishmania donovani complex. It is usually fatal if untreated. The annual incidence and prevalence of visceral leishmaniasis cases worldwide is -0.5 million and ~2.5 million, respectively. The current available chemotherapeutic drugs have the disadvantages of serious side effects, progressive development of resistance, lack of oral formulation or high cost. And co-infection of AIDS and VL makes the treatment more difficult. So, new chemotherapeutic drugs are needed to supplement or replace currently available drugs in use. Our previous study indicated that L. donovani promastigotes were highly inhibited by dihydroartemisinin in vitro. The aim of this study is to observe effect of dihydroartemisinin on experimental murine visceral leishmaniasis induced by L. donovani. Three groups of golden hamsters, including two experimental groups and one control group (n=5), were injected with L. donovani amastigotes through intraperitoneal route. Two weeks later mice of the two experimental groups were administered with dihydroartemisinin orally at the dose of 25mg/kg/d and 50mg/kg/d respectively. Drug was given once a day for 14 days. We determined the parasite burden in spleen and liver of every group by both limiting dilution method and Stauber's method. Statistical significance (P<0.05) was analyzed by Student's t-test. The tissue parasite burden of 4/5 dihydroartemisinin treated mice was obviously decreased than controls. Inhibition rate of dihydroartemisinin at the dose of 25mg/kg/d was 72.46% in spleen and 80.92% in liver and that of dihydroartemisinin at the dose of 50mg/kg/d was 75.45% in spleen and 80.92% in liver by limiting dilutionmethod. The data from Stauber's method were well consistent with those from limiting dilution method. There was no significance between the effect of low-dose experimental group and that of high-dose one. It was first identified in our study that dihydroartemisinin had potent activity against experimental murine visceral leishmaniasis. Moreover, dihydroartemisinin has the advantages of oral administration, low side-effect and low cost. That is very important for treatment of visceral leishmaniasis of which more than 90% cases are in poor developing countries. The results of our research could found a basis for further studies on treatment of human VL and canine VL with dihydroartemisinin, and could found a new way for treatment and prevention ofVL.According to geographic distribution and source of infection, the visceral leishmaniasis in China may be divided into plain type, hill type and desert type. Different types display differences in the age, effect of treatment, relapse and reservoir host. Recently the number of visceral leishmaniasis cases in hill foci of China went up again. And studies for prevention and treatment of this disease are still urgent. Leishmania homologue of receptors for activated protein kinase C (LACK) was recently discovered in Leishmania major. This protein is a highly conserved protein among related Leishmainia species and a promising candidate for the development of a vaccine against human leishmaniasis. In this study the LACK gene of Leishmainia donovani (L d.) SD1 from plain foci and L d. XJ771 from desert foci were amplified by RT-PCR. The results of sequencing showed that both of the 2 fragments were 942bp in length. Comparisons of the two nucleotide sequences with that of other Leishmania spp. in Gene Bank indicated that the identities were more than 97%, and the identities of the nucleotide sequences of LACK gene of the three L.d isolates from plain, desert and hill foci of China respectively were more than 95%. Comparisons of the deduced amino-acid sequences with that of other Leishmania spp. in Gene Bank indicated that the identities were more than 97%, and the identities of the deduced amino-acid sequences of LACK gene of the three L. d. isolates from plain, desert and hill foci of Chinarespectively were more than 95%. High identities among the nucleotide sequences of LACK gene of the three L d. isolates from three different foci of China existed. Considering that most visceral leishmaniasis cases in China were from hill foci, the LACK gene of the L d. isolate from hill foci kept in our lab was cloned into eukaryotic expressed plasmid pcDNA3.1(+) to construct recombinant plasmid. This recombinant plasmid was then transfected into the eukaryotic cell COS-7 and the expression of LACK gene in eukaryotic cell was detected by RT-PCR and immunofluorescent staining. Both RT-PCR and immunofluorescent staining of recombinant plasmid transfected COS-7 showed positive reaction. So, we concluded that the recombinant plasmid pcDNA3-LACK could express LACK protein in eukaryotic cell COS-7, which could found a basis of developing a new genetic Leishmania vaccine of China.The last part of this report included the research on adherence-conferring factor of shiga toxigenic Escherichia coli (STEC). Though Shiga toxin is a very important agent for the clinical and pathologic manifestations of STEC infection, some toxic factors, especially colony and adherence conferring factors, are the key determinant agents for the disease. In this study, the entire DNA sequence of a 5.2kb-sized Hind III fragment from LEE negative STEC 0113: H7 isolate 98NK2 was obtained through southern hybridization, cloning, dot-hybridization and DNA sequencing. The nucleotide sequence of a 2088bp-sized open reading frame designated Ihaomin this fragment had 93% identities with that of ihaoi57, an putative adhesin from LEE positive STEC 0157: H21 isolate, and the identities of the two deduced amino-acid sequences were 91%. The nucleotide sequence of iha, an anticipated adherence-conferring factor, from LEE negative STEC 0113: H7 isolate was first reported in this study, which could provide an important theoretical basis for further studies on its molecular mechanism of pathogenesis.