Entomological monitoring of Leishmania infection in leishmaniasis endemic areas offers epidemiologic advantages for predicting the risk and expansion of the disease, as well as evaluation of the effectiveness of control programs. In this study, we developed a highly sensitive loop-mediated isothermal amplification (LAMP) method for the mass screening of sand flies for Leishmania infection based on the 18S rRNA gene. The LAMP technique could detect 0.01 parasites, which was more sensitive than classical PCR. The method was robust and could amplify the target DNA within 1 hr from a crude sand fly template without DNA purification. Amplicon

The surveillance of prevalent Leishmania and sand fly species in endemic areas is important for prediction of the risk and expansion of leishmaniasis. In this study, we developed a polymerase chain reaction (PCR)-based method for detection of Leishmania minicircle DNA within individual sand flies. Using this method, we detected minicircle DNA in 6 (3.3%) of 183 sand flies, while 5 (3.5%) of 143 were positive for Leishmania promastigotes in the same areas by microscopic examination. The species were identified as Leishmania (Leishmania) mexicana by nucleotide sequencing of the cytochrome b gene. Additionally, all the Leishmania-positive sand flies were identified as Lutzomyia ayacuchensis by the restriction enzyme digestion of the PCR-amplified 18S ribosomal RNA gene fragments. Since this combined method is relatively easy and can process a large number of samples, it will be a powerful tool for the rapid identification of prevalent sand fly and Leishmania species as well as monitoring the infection rate in sand fly populations in endemic areas.

S U M M A R YThe Cytochrome b (Cyt b) gene has proved to be useful for identification and classification of many mammals and plants. In order to evaluate the utility of this gene for discrimination of Leishmania parasites as well as for exploring their phylogenetic relationships, we determined the nucleotide sequences of the Cyt b gene from 13 human-infecting Leishmania species (14 strains) from the New and Old Worlds. The Cyt b genes, approximately 1080 base pairs, were found to be A/T rich, and their 5k terminal-editing regions were highly conserved. The nucleotide sequence variation among them was enough to discriminate parasite species; 245 nucleotide positions were polymorphic and 190 positions were parsimony informative. The phylogenetic relationships based on this gene, showed good agreement with the classification of Lainson & Shaw (1987) ) tropica complex and the placement of L. tarentolae in another genus. These data show that the Cyt b gene is useful for phylogenetic study of Leishmania parasites.

This study aimed to precisely discriminate Fasciola spp. based on DNA sequences of nuclear internal transcribed spacer 1 (ITS1) and mitochondrial nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 1 (nad1) gene. We collected 150 adult flukes from the bile ducts of cattle, buffaloes, sheep, and goats from six different regions of Bangladesh. Spermatogenic status was determined by analyzing stained seminal vesicles. The ITS1 types were analyzed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The nad1 haplotypes were identified based on PCR and direct sequencing and analyzed phylogenetically by comparing with nad1 haplotypes of Fasciola spp. from other Asian countries. Of the 127 aspermic flukes, 98 were identified as Fg type in ITS1, whereas 29 were identified as Fh/Fg type, indicating a combination of ITS1 sequences of Fasciola hepatica and Fasciola gigantica. All the 127 aspermic flukes showed Fsp-NDI-Bd11 in nad1 haplotype with nucleotide sequences identical to aspermic Fasciola sp. from Asian countries. Further, 20 spermic flukes were identified as F. gigantica based on their spermatogenic status and Fg type in ITS1. F. gigantica population was thought to be introduced into Bangladesh considerably earlier than the aspermic Fasciola sp. because 11 haplotypes with high haplotype diversity were detected from the F. gigantica population. However, three flukes from Bangladesh could not be precisely identified, because their spermatogenic status, ITS1 types, and nad1 haplotypes were ambiguous. Therefore, developing a robust method to distinguish aspermic Fasciola sp. from other Fasciola species is necessary in the future.

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Alveolar echinococcosis, which is due to the massive growth of larval Echinococcus multilocularis, is a life-threatening parasitic zoonosis distributed widely across the northern hemisphere. Commercially available chemotherapeutic compounds have parasitostatic but not parasitocidal effects. Parasitic organisms use various energy metabolic pathways that differ greatly from those of their hosts and therefore could be promising targets for chemotherapy. The aim of this study was to characterize the mitochondrial respiratory chain of E. multilocularis, with the eventual goal of developing novel antiechinococcal compounds. Enzymatic analyses using enriched mitochondrial fractions from E. multilocularis protoscoleces revealed that the mitochondria exhibited NADH-fumarate reductase activity as the predominant enzyme activity, suggesting that the mitochondrial respiratory system of the parasite is highly adapted to anaerobic environments. High-performance liquid chromatography-mass spectrometry revealed that the primary quinone of the parasite mitochondria was rhodoquinone-10, which is commonly used as an electron mediator in anaerobic respiration by the NADH-fumarate reductase system of other eukaryotes. This also suggests that the mitochondria of E. multilocularis protoscoleces possess an anaerobic respiratory chain in which complex II of the parasite functions as a rhodoquinol-fumarate reductase. Furthermore, in vitro treatment assays using respiratory chain inhibitors against the NADH-quinone reductase activity of mitochondrial complex I demonstrated that they had a potent ability to kill protoscoleces. These results suggest that the mitochondrial respiratory chain of the parasite is a promising target for chemotherapy of alveolar echinococcosis.Echinococcosis is a near-cosmopolitan zoonosis caused by helminthic parasites belonging to the genus Echinococcus (family Taeniidae) (18). The life cycle of Echinococcus spp. includes an egg-producing adult stage in the definitive hosts and a larval stage in intermediate hosts including humans. The larval stage of the parasite produces a large number of infective protoscoleces that develop to adult worms after being ingested by the definitive host, or they produce a new parasite mass when liberated inside the intermediate host, causing metastases of the parasite lesions. The two major species of medical and public health importance are Echinococcus granulosus and E. multilocularis, which cause cystic echinococcosis and alveolar echinococcosis (AE), respectively.Human AE is a life-threatening disease, and without careful clinical management, it has a high fatality rate and poor prognosis. Humans acquire AE infection by ingesting eggs from adult parasitic worms. Early diagnosis and treatment (mainly by radical surgery) of human AE are difficult because the disease progresses slowly and usually takes more than several years before clinical symptoms become apparent. An efficient chemotherapeutic compound is still not available. The first choice for the chemotherapy of AE is benz...

Mitochondrial DNA variation in the cytochrome b (cyt b) gene and the control region was examined in the red fox Vulpes vulpes from Japan, with special focus on the population divergence between Hokkaido and northern Honshu. Resultant haplotypes from Hokkaido were subdivided into two distinct groups (I and II), with an average genetic distance of 0.027 for cyt b. Divergence time is roughly estimated to be 1-2 million years ago, given that the conventional divergence rate of the mammalian cyt b gene is 2% per million years. Notably, Group II was only found in Hokkaido, whereas Group I comprised haplotypes from Honshu, Kyushu (Japan), eastern Russia, and Europe, as indicated by a comparison of our own data to the literature. On the other hand, judging from constructed trees, Group I haplotypes from Hokkaido appeared to differ from those from other parts of Japan, i.e., Honshu and Kyushu. This implies that Blakiston's Line, which demarcates the boundary between Hokkaido and Honshu, has been an effective barrier and has allowed the structuring of genetic variation in maternal lineages. Thus, these results suggest that the Hokkaido population, which is sometimes referred to as the distinct subspecies V. v. schrencki, has its own genetic background with multiple migration events and differs from the parapatric subspecies V. v. japonica found in Honshu and Kyushu.