The development of oocytes in the ovary of a parthenogenetic tick, Haemaphysalis longicornis.

Haemaphysalis longicornis is an important vector of various pathogens in domestic animals and humans. The tick is a unique species with bisexual and parthenogenetic races. Although mating induces oocyte development, it is possible in the parthenogenetic race to complete oogenesis without copulation. Here we examined the developmental process of oocytes from unfed to the oviposition period in parthenogenetic H. longicornis. We classified the developmental stages of oocytes into five stages: stage I, germinal vesicle occupies more than half of the cytoplasm; stage II, germinal vesicle occupies less than half of the cytoplasm; stage III, germinal vesicle migrates from the center in the oocyte to the vicinity of the pedicel cells; stage IV, the cytoplasm is filled with yolk granules of various sizes; stage V, the cytoplasm is occupied by large yolk granules. Oocytes at the unfed period were undeveloped and classified as stage I. Stage I and II oocytes were observed at the rapid feeding period, indicating that oocyte development began after the initiation of blood feeding. All developmental stages of oocytes were observed at the pre-oviposition period. At 10 days after the beginning of the oviposition period, the ratios of stage I and II oocytes were higher than those of the previous period, suggesting that the ovarian development and activity may be continuing. Based on these findings, we propose classification criteria for the oocyte development in the parthenogenetic H. longicornis. The criteria will be useful for understanding the mechanisms of tick reproduction and transovarial transmission of pathogens.

Expression analysis of autophagy-related genes in the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous arthropods with unique life cycles characterized by relatively short feeding periods and long non-feeding periods. They ambush a suitable host animal while staying in a pasture without any food source for up to several months. To understand the molecular mechanisms underlying their exceptional viability, we focused on autophagy, a proteolysis system via the lysosomes that is induced by starvation in eukaryotes. We hypothesized that starved conditions facilitate autophagy during host-seeking periods in the life cycle of the tick. To date, homologues of five autophagy-related (ATG) genes, ATG3, ATG4, ATG6, ATG8, and ATG12, have been identified from the hard tick Haemaphysalis longicornis. We showed previously that the mRNA levels of H. longicornis ATG (HlATG) genes were higher during the non-feeding period than the feeding period in the nymphal to adult stages. In addition, the expressions of HlATG3, HlATG4, HlATG8 and HlATG12 were highest in the egg compared to the other developmental stages in the same tick. In the present study, we used real-time polymerase chain reaction to examine the expression profiles of HlATG genes in the embryonic stage, larval to nymphal stages, and in internal organs of female ticks. We found that the HlATG genes were expressed at the highest levels in developing eggs on day 0 after oviposition. The levels of HlATG4 and HlATG8 were higher during the non-feeding period than the feeding period in the larval to nymphal stages. In the adults, the unfed condition appeared to be associated with the increased expression of HlATG genes in the fat body and midgut, which are nutrient storage organs; however, the expression patterns of HlATG genes varied in other organs. These results suggest that an up-regulation of HlATG genes is not always induced in different organs of unfed female ticks. Taken together, our findings raise the new possibility that HlATG genes play distinct biological roles in eggs, unfed ticks, engorged ticks (metamorphosis), and in each organ.

Epidemiological survey of Theileria parasite infection of cattle in Northeast China by allele-specific PCR.

An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.

Cloning and characterization of the autophagy-related gene 6 from the hard tick, Haemaphysalis longicornis.

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.

Identification of the cross-reactive and species-specific antigens between Neospora caninum and Toxoplasma gondii tachyzoites by a proteomics approach.

The characterization of the cross-reactive and species-specific antigens of Neospora caninum and Toxoplasma gondii is important in the exploration to determine the common mechanisms of parasite-host interaction and to improve the serological diagnosis; it is also useful for the selection of the cross-reactive antigens that could be used in the development of vaccines or drugs for controlling the diseases caused by these two parasites. In this study, cross-reactive and species-specific antigens between N. caninum and T. gondii tachyzoites were comprehensively investigated using a proteomics approach with the application of two-dimensional gel electrophoresis, immunoblot analysis, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS), and MALDI-TOF/TOF-MS analysis. Immunoblotting and mass spectrometry analysis revealed that at least 42 individual protein spots of N. caninum were reacted with the anti-N. caninum serum, among which at least 18 protein spots were cross-reacted with the anti-T. gondii serum. Moreover, at least 31 protein spots of T. gondii were reacted with the anti-T. gondii serum, among which at least 19 protein spots were cross-reacted with the anti-N. caninum serum. Furthermore, some new specific proteins were also identified in the N. caninum protein profile by searching Toxoplasma sequences or sequences from other organisms. This study substantiates the usefulness of proteomics in the immunoscreening of the cross-reactive or species-specific antigens of both parasites. In addition, the present study showed that there was significant homology in the antigenic proteome profiles between the two parasites. These observations have implications for the design of multicomponent common vaccines against both parasite infections.

Construction and analysis of full-length cDNA library of Cryptosporidium parvum.

A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies.

Identification and characterization of a novel secreted antigen 1 of Babesia microti and evaluation of its potential use in enzyme-linked immunosorbent assay and immunochromatographic test.

Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir.

The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis.

A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41-79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.