The effect of normal human serum on the mouse trypanosome Trypanosoma musculi in vitro and in vivo.

Trypanosoma musculi, a common blood flagellate found in mice, is similar in morphology and life cycle to the rat trypanosome T. lewisi. Both species belong to the subgenus Herpetosoma, and as T. lewisi has recently been shown to be a zoonotic pathogen, there is concern that T. musculi could also be potentially infective to humans. To test this hypothesis, a well-established method, the normal human serum (NHS) incubation test, was carried out which distinguishes human and non-human infective trypanosomes. We found that T. musculi could grow in 0.31% NHS in vitro, and even kept their infectivity to mice after incubation with 10% NHS for 24 h. In in vivo experiments, T. musculi were only slightly affected by NHS injection, confirming that it was less sensitive to the NHS than T. b. brucei, but more sensitive than T. lewisi. This resistance probably does not rely on a restricted uptake of ApoL-1. Due to this partial resistance, we cannot definitively confirm that T. musculi has the potential for infection to humans. As resistance is less than that of T. lewisi, our data suggest that it is unlikely to be a zoonotic pathogen although we would advise caution in the case of immunocompromised people such as AIDS and cancer patients.

PCR-based identification of Trypanosoma lewisi and Trypanosoma musculi using maxicircle kinetoplast DNA.

Trypanosoma lewisi, transmitted by rat fleas, is a widespread pathogen specific to rats with records of human infection cases. Its closely related species with global distribution, Trypanosoma musculi, is transmitted between mice by ingestion of infected fleas. These trypanosomes are of similar morphology, making it difficult to distinguish them by microscopy. In this study, we have developed a rapid, sensitive and reliable PCR method for the diagnosis of T. lewisi and T. musculi. The T. lewisi-specific amplicons were not produced by other Trypanosoma, such as T. musculi, T. brucei complex or T. cruzi, neither by an outgroup of Leishmania amazonensis. The detection limits of the three pairs of T. lewisi-specific primers were 50ng, 1ng and 10ng of total DNA, respectively. The primers designed for T. musculi primers showed specifically that amplicon strictly in T. musculi and their detection limits were 10ng and 1ng of total DNA. To simplify the detection process, we managed to apply our method directly on tail blood samples without complicated DNA purification. In conclusion, PCR with our primers could be a highly sensitive, specific protocol to detect and distinguish T. lewisi and T. musculi from other trypanosomes.

Current status of Clonorchis sinensis and clonorchiasis in China.

The oriental liver fluke, Clonorchis sinensis, a pathogen causing clonorchiasis, is of major socio-economic importance in East Asia, including China, Korea and Vietnam. This parasite is now recognized as a biocarcinogen strongly linked to cholangiocarcinoma in humans. Here, we describe the status of clonorchiasis in China, where it has been estimated that more than 15 million patients are affected. This paper also summarizes the major advances in the field of clonorchiasis research during last decade, including diagnosis techniques, pathogenesis and genome/transcriptome/proteome studies in the last years. We strongly hope that our work can stimulate the governments of the countries or regions where clonorchiasis is endemic to pay more attention to this disease and establish related guidelines to prevent and control it.

[Pathogen Identification in an Imported Case of Cutaneous Leishmaniasis].

In this study, immune and molecular biological methods were used to identify the pathogen in a blood sample from a patient with dermatosis. Venous blood was collected and tested with Leish rK39 dipsticks. The lesion sample was collected and fixed in 75% ethanol, and DNA was extracted. The internal transcribed spacer 1 of rDNA and N-acetylglucosamine-1-phosphate transferase of Leishmania were amplified with PCR using primers LITSR-L5.8S and NAGTL1s-NAGTL4, respectively. The amplified products were sequenced and analyzed by BLAST. Weakly positive results were obtained for the gold-labeled Leish rK39 dipstick serological test. PCR resulted in products of 404 bp and 1 405 bp with primers LITSR-L5.8S and NAGTL1-NAGTL4, respectively. Both were 99.7% homologous to the corresponding sequence of Leishmania major. The accession number of the two sequences were KU975160 and KX150476. The case of dermatosis is diagnosed as imported cutaneous leishmaniasis and the pathogen is L. major.

[Separation of bile acids by capillary zone electrophoresis].

AIM: To develop a method for separating the major bile acids by capillary zone electrophoresis (CZE). METHODS: The effect of different separations, such as the compose, pH and the concentration of buffer, on the electro-osmotic flow (EOF), the migration time and resolution of 8 bile acids in this system were studied. The general trends in migration time could be correlated to the pH and concentration of the buffer. The effect of organic reagent on EOF and migration time were also investigated. By addition of methanol, the EOF went smaller than before, and better resolution was achieved. The experimental results showed that optimum separation was achieved under the following condition: buffer composition of 126 mmol.L-1 disodium tetraborate, 43 mmol.L-1 disodium hydrogenphosphate, 18% methanol; temperature 30 degrees C; voltage 30 kV; total length of capillary 570 mm and 500 mm from injection end; ultraviolet detection at 200 nm; pressure injection 5 kPa for 8 s. RESULTS: Eight kinds of bile acid had been separated by CZE with only one injection. The method was used to analyse the contents of bile acids from different kinds of bear biles, the recovery was 89%-107%. CONCLUSION: This method is simple and rapid, and can be used to determine the content of bile acids in bear biles. The calibration curve showed good linearity for eight bile acids in the concentration range of 4-60 mg.mL-1 (gamma > 0.9954). The total time for seperation and determination was within 25 min.

[Separation of different phosphatidyl cholines by reversed-phase ion-pair high performance liquid chromatography].

A reversed-phase ion-pair high performance liquid chromatographic (RP-IP-HPLC) method for the separation of different phosphatidyl cholines (PC) was established. The optimized conditions were a PERKIN-ELMER/HS-5 C18 column (150 mm x 4.6 mm i.d., 5 microns), isocratic elution with mobile phase of methanol-acetonitrile-water (70:22:8, volume ratio) containing 15 mmol/L tetramethyl ammonium phosphate (TMAP) at pH 7.0, flow rate of 2.0 mL/min and UV detection at 208 nm. Seven kinds of PC could be separated with this method.