Zhao-Rong Lun

Clonorchiasis: a key foodborne zoonosis in China

The oriental liverfluke, Clonorchis sinensis, is of major socioeconomic importance in parts of Asia, including China, Japan, Korea, Taiwan, and Vietnam. The parasite is transmitted via snails to freshwater fish, and then to human beings and other piscivorous mammals, and causes substantial clinical or subclinical disease, known as clonorchiasis. There is considerable evidence for an aetiological relation between clonorchiasis and cholangiocarcinoma in human beings. It is estimated that about 35 million people are infected globally, of whom approximately 15 million are in China. Although very little information from China has been published in the English language, recent analyses of epidemiological data sets suggest that clonorchiasis is having an increased human-health impact due to the greater consumption of raw freshwater fish. To gain an improved insight into clonorchiasis in China, this review provides a background on the parasite and its life cycle, summarises key aspects regarding the pathogenesis, diagnosis, and treatment of clonorchiasis, describes the geographic distribution and prevalence of clonorchiasis, and makes some recommendations for future research and the control of this important disease.

Immunization with recombinant beta-tubulin from Trypanosoma evansi induced protection against T. evansi, T. equiperdum and T. b. brucei infection in mice

The beta‐tubulin gene of Trypanosoma evansi (STIB 806) was cloned and expressed in Escherichia coli. The predicted amino acid sequence of T. evansi beta‐tubulin shows 100%, 99·8%, 99·1%, and 98·6% homology with T. equiperdum, T. b. brucei, T. cruzi and T. danilewskyi, respectively, but is diverse from that of T. cyclops, showing only 51·6% of homology. Recombinant beta‐tubulin was expressed as inclusion bodies in E. coli. It was purified and renatured for immunological studies. Mice immunized with the renatured recombinant beta‐tubulin were protected from lethal challenge with T. evansi STIB 806, T. equiperdum STIB 818 and T. b. brucei STIB 940, showing 83·3%, 70% and 76·7% protection, respectively. Serum collected from the rabbit immunized with recombinant beta‐tubulin inhibited the growth of T. evansi, T. equiperdum and T. b. brucei in vitro. Serum from mice and rabbits immunized with recombinant beta‐tubulin recognized only T. evansi beta‐tubulin and not mouse beta‐tubulin. The results of this study demonstrated that the recombinant T. evansi beta‐tubulin is a potential candidate for the development of a vaccine to prevent animal trypanosomiasis caused by these three trypanosome species.

Genetic evidence for the existence of sibling species within Contracaecum rudolphii (Hartwich, 1964) and the validity of Contracaecum septentrionale (Kreis, 1955) (Nematoda: Anisakidae)

Specimens of Contracaecum rudolphii sensu lato (s.l.) (Nematoda: Anisakidae) from Phalacrocorax carbo sinensis from northeastern and central Italy were characterised genetically and compared with those from Phalacrocorax aristotelis from Galician coasts, Spain (identified as C. rudolphii A by multilocus enzyme electrophoresis) and with specimens of C. septentrionale from Alca torda from the Galician coasts, Spain. The first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of ribosomal DNA (rDNA) were amplified by polymerase chain reaction (PCR) from individual nematodes and the amplicons subjected to single-strand conformation polymorphism (SSCP) analysis and/or sequencing. For each ITS region, C. septentrionale specimens were distinct from those of C. rudolphii (s.l.) and C. rudolphii A based on SSCP profiles and ITS sequences. Some specimens of C. rudolphii (s.l.) had the same SSCP profiles and ITS sequences as C. rudolphii A, whereas the others had distinct SSCP profiles and ITS sequences and were suggested to represent C. rudolphii B based on host and geographical origins and genetic similarity to C. rudolphii A. While no length or nucleotide variation in the ITS-1 and ITS-2 sequences was detected within each taxon, nucleotide differences of 1.8–5.5% (ITS-1) and 5.1–12.2% (ITS-2) were detected among them. The results support the hypothesis that C. rudolphii represents a complex of at least two sibling species and provide support for the validity of C. septentrionale as a separate species. The definition of genetic markers in the ITS rDNA provides opportunities for investigating the life cycles, transmission patterns and ecology of the anisakid nematodes studied herein.

The occurrence of Toxocara malaysiensis in cats in China, confirmed by sequence-based analyses of ribosomal DNA

Non-isotopic polymerase chain reaction (PCR)-based single-strand conformation polymorphism and sequence analyses of the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA) were utilized to genetically characterise ascaridoids from dogs and cats from China by comparison with those from other countries. The study showed that Toxocara canis, Toxocara cati, and Toxascaris leonina from China were genetically the same as those from other geographical origins. Specimens from cats from Guangzhou, China, which were morphologically consistent with Toxocara malaysiensis, were the same genetically as those from Malaysia, with the exception of a polymorphism in the ITS-2 but no unequivocal sequence difference. This is the first report of T. malaysiensis in cats outside of Malaysia (from where it was originally described), supporting the proposal that this species has a broader geographical distribution. The molecular approach employed provides a powerful tool for elucidating the biology, epidemiology, and zoonotic significance of T. malaysiensis.

Symbiosis of Mycoplasma hominis in Trichomonas vaginalis may link metronidazole resistance in vitro

Fourteen of 28 Trichomonas vaginalis isolates collected from patients in Guangzhou, China from 2003 to 2004 were found to be naturally infected with Mycoplasma hominis, as determined by PCR using specific primers. In vitro metronidazole sensitivity assay of the 28 isolates revealed four displaying low susceptibility [minimum lethal concentration (MLC)=∼13–25xa0μg/ml] and another four displaying high resistance (MLC=50–100xa0μg/ml). The overwhelming majority of these resistant isolates (7/8) were mycoplasma-infected. The mean of MLCs of mycoplasma-infected isolates is ∼10-fold higher than the mean of noninfected isolates (p=0.029). Sequence analyses of PCR-amplified small subunit–large subunit rRNA interspacer regions (ITS1/5.8S/ITS2) revealed that 23 of the 28 samples are identical, the remaining five being separable into two groups, each with a single point mutation. These internal transcribed spacer sequence variants are associated neither with mycoplasma infection nor with drug resistance. In contrast, random amplified polymorphic DNA analyses of DNAs using 10 different primers showed that the drug-resistant isolates are clustered together in association with mycoplasma infection, albeit more loosely. Taken together, the results obtained from this study suggest that in vitro metronidazole resistance of T. vaginalis is related to mycoplasma infection of this protozoan.

Specific PCR assays for the identification of common anisakid nematodes with zoonotic potential

Based on the sequences of the internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) for six taxa of anisakids, namely, Anisakis simplex (s.s.), Anisakis typica, Anisakis pegreffii, Hysterothylacium aduncum, Hysterothylacium sp, and Contracaccum osculatum C, specific primers were designed in the ITS-1 and/or ITS-2 for each of the six anisakid taxa. These specific primers were used to develop polymerase chain reaction (PCR) tools for the identification of these anisakid taxa of sea fish by amplifying partial ITS-1 and/or ITS-2 of rDNA from anisakid nematodes. This approach allowed their specific identification, with no amplicons being amplified from heterogeneous DNA samples, and sequencing confirmed the identity of the DNA fragments amplified. The minimum amounts of DNA detectable using the PCR assays were 0.5–1xa0ng. These PCR tools were then applied to ascertain the specific identity of 143 anisakid larval samples collected from fish in China, Canada, Thailand, and Indonesia, and these anisakid samples were identified to represent one of the six anisakid taxa. These PCR assays based on ITS sequences should provide useful molecular tools for the accurate identification and molecular epidemiological investigations of anisakid infections in humans and fish.

Seroprevalence of Toxoplasma gondii Infection in Stray and Household Cats in Guangzhou, China

The prevalence of anti‐Toxoplasma gondii specific IgG in stray and household cats in Guangzhou, China was determined by ELISA on serum samples from 206 cats (117 strays and 89 households) and the overall infection rate was 25.24%. The infection rate in stray cats (30.77%) was significantly higher (Pu2003<u20030.05) than in household cats (17.98%). The rate of infection between male and female cats of both groups was not significantly different (Pu2003≥u20030.05), 28.13% versus 32.61% for male and female in stray cats, respectively, and 18% versus 17.95% in household cats. The present investigation demonstrated that the prevalence of T. gondii infection in cats in Guangzhou was high, especially in stray cats, which are probably the main source of T. gondii infection in this area. Integrated control strategies and measures should be implemented to prevent and control T. gondii infection in both stray and household cats, which will have significant implications for the control of human infection with T. gondii.

Tandem repeat protein as potential diagnostic antigen for Trypanosoma evansi infection

Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48xa0days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.

Atypical human trypanosomiasis: a neglected disease or just an unlucky accident?

Several cases of human infections with animal trypanosomes such as Trypanosoma evansi, Trypanosoma lewisi and Trypanosoma congolense have been reported; this raises the question as to whether they have the potential to become new diseases of humans or whether they simply represent a biological accident.

Development and evaluation of loop-mediated isothermal amplification (LAMP) for rapid detection of Clonorchis sinensis from its first intermediate hosts, freshwater snails

Clonorchiasis, caused by Clonorchis sinensis, is a key foodborne zoonosis, which is mainly found in China, Korea and Vietnam. Detection of this parasite from the second intermediate host, the freshwater fish is the common method for epidemiological surveys of this parasite, but is time consuming, labour intensive and easily leads to misdiagnosis. In this study, we have developed a rapid, sensitive and reliable molecular method for the diagnosis of C. sinensis from its first intermediate hosts, freshwater snails, based on a loop-mediated isothermal amplification (LAMP) method. The specific amplified fragment from genomic DNA of C. sinensis did not cross-react with those from other relevant trematodes and a range of hosts (freshwater fish, shrimps and snails) of C. sinensis living in similar environments. The detection limit of the LAMP method was as low as 10 fg which was 1000 times more sensitive than conventional PCR, which was also demonstrated by successful application to field samples. These results show that the LAMP method is a more sensitive tool than conventional PCR for the detection of C. sinensis infection in the first intermediate hosts and, due to a simpler protocol, is an ideal molecular method for field-based epidemiological surveys of this parasite.