Molecular cloning of two Haemaphysalis longicornis cathepsin L-like cysteine proteinase genes.

Immunological protection of mammalian hosts against tick infestation has been proposed as the most sustainable alternative tick control method to the current use of acaricides which has several limitations. The success of this method is dependent on the identification of key molecules for use as tick vaccine antigens. Proteolytic enzymes are involved in a wide range of cellular processes in eukaryotes such as development regulation and nutrition, thus they can be considered as good target antigens for a tick vaccine. In the present study we used primers designed based on the consensus amino acid motifs flanking the conserved active sites C25 and N175 present in all papain-like cysteine proteinases to amplify by polymerase chain reaction, sequence and characterize two Haemaphysalis longicornis tick cysteine proteinase genes. Based on the nucleotide and deduced amino acid sequences, both genes were identified as members of the cysteine proteinase gene family by presence in their sequences of consensus motifs flanking the conserved active sites C25, H150 and N175 that are present in all papain-like cysteine proteinases. Both genes are about 1.2 kb in size and show high sequence homology predominantly to invertebrate cathepsin L-like cysteine proteinases.     

Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.     

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.     

Development of Babesia ovata in the midgut of the tick, Haemaphysalis longicornis

Studies were made on the development of Babesia ovata in the midgut of the nymphal tick vector, Haemaphysalis longicornis. In 12 hr post-repletion, merozoites were observed outside of erythrocytes infected with B. ovata in the contents of the midgut of the tick. After that, these merozoites were transformed into ring-forms which were comparatively large ring 2-3 microns in diameter. Within 48-72 hr post-repletion, ring-form protozoa developed into spherical form 4-5 microns in diameter. Within 3-4 days post-repletion, fission-forms were transformed into fission-bodies 2-3 microns in diameter. Within 4-6 days post-repletion, fission-bodies developed into bizarre-forms 6-7 microns in diameter. At this time, elongated form protozoa which were considered as microgametes, 6-8 microns in length, are also seen. Within 6-8 days post-repletion, round-formed protozoa which were considered as zygotes in 9-10 microns in diameter were observed in the gut. About 10 days after repletion, those round-formed protozoa were transformed into vermicule-formed and round-formed protozoa, 13-15 microns in length, appeared again in the gut epithelial cells.     

High incidence of rickettsiosis correlated to prevalence of Rickettsia japonica among Haemaphysalis longicornis tick

Endemic spotted fever group rickettsiosis was reported in Shimane Prefecture, Japan. From an analysis of 14 clinical cases found in the endemic area, the infectious agent of spotted fever group rickettsiosis was identified as Rickettsia japonica. In this study, we also found that Rickettsia japonica was highly infected with the vector tick, Haemaphysalis longicornis, in the endemic area. These findings suggest that the high incidence of rickettsiosis in Shimane Prefecture can be explained by the high prevalence of Rickettsia japonica among Haemaphysalis longicornis ticks.     

Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

Molecular cloning and sequence analysis of cDNAs encoding for Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus actins

The nucleotide and deduced amino acid sequences of the actins from ticks, Boophilus microplus, Haemaphysalis longicornis and Rhipicephalus appendiculatus, have been determined. Nucleotide sequence analysis showed open reading frames of 1128-nucleotide-long encoding proteins of 376 amino acids with a predicted molecular weight of 41.82 kDa each. Comparison between the nucleic acid and deduced amino acid sequences as well as structural and phylogenetic analyses of these genes confirmed the high similarity among actins from ticks in comparison to other species.     

镰形扇头蜱肌钙蛋白I基因的克隆及其分布

本试验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的EST中筛选了1个含polyA尾的基因序列,经5LRACE方法得到该基因全长序列.经同源性比较,该基因预测的氨基酸序列与长角血蜱肌钙蛋白I(GI14041807)的同源性为84.47%,肌动蛋白结合位点位于147-167氨基酸处,且与长角血蜱肌钙蛋白I肌动蛋白结合位点完全相同,表明该基因是镰形扇头蜱肌钙蛋白I基因.以镰形扇头蜱基因组DNA为模板扩增到编码肌钙蛋白I的基因组序列.序列分析表明该序列不含内含子.RT-PCR分析表明该基因在镰形扇头蜱的卵及其幼蜱、若蜱、成蜱的壳、唾液腺和肠均有表达.     

Molecular characterization of a myosin alkali light chain-like protein, a "concealed" antigen from the hard tick Haemaphysalis qinghaiensis

There should be some differences between antibodies generated by feeding ticks on animals and those derived by immunizing animals with tick extracts. Here, we found serum collected from sheep immunized with Haemaphysalis qinghaiensis salivary gland extracts could detect two more protein bands with molecular weights of 22 and 37 kDa (P22 and P37) on Western blots of extracts of tick salivary glands than serum from tick infected animals. Rabbit anti-H. qinghaiensis differential protein immune serum was then generated from P22 and P37 and was used to immunoscreen a cDNA library constructed from salivary glands, Malpighian tubules and ovaries of partially engorged H. qinghaiensis. A cDNA contains an open reading frame of 483 bp that codes for 160 amino acid residues with a coding capacity of 18 kDa was cloned and designated Hq02. Expression analysis by RT-PCR showed that this gene is expressed in salivary glands, midguts, other organs and different developmental stages of H. qinghaiensis. The predicted amino acid sequence of the Hq02 gene had high homology to some known myosin alkali light chain (MLC) proteins. A fusion protein consisting of 130 amino acids of Hq02 protein and 335 amino acids of T7 gene 10 protein was expressed in Escherichia coli and used to immunize sheep. Western blot showed that only rabbit anti-H. qinghaiensis differential protein immune serum could recognize the expressed Hq02 protein, while rabbit anti-H. qinghaiensis saliva immune could not. This proved Hq02 protein was a "concealed" antigen. Immunization with the recombinant Hq02 conferred a 21.8% reduction of engorgement weight for adult female ticks that fed on the immunized sheep. This is the first report of tick myosin alkali light chain and the function of this protein is discussed.     

长角血蜱丝氨酸蛋白酶抑制剂基因的原核表达

根据GenBank中的长角血蜱丝氨酸蛋白酶抑制荆(Haemaphysalislongicornis serine proteinase inhibitor-2,HLS2)基因序列设计特异性引物,以长角血蜱饥饿成蜱总RNA为模板,经RT-PCR扩增出1164 bp的HLS2DNA片段.将其与pET-30a载体连接,构建pET-30a-HLS2表达载体,转化JM109大肠杆菌,筛选阳性克隆,经双酶切鉴定及测序分析后转化到E.coli BL21(DE3)表达菌株中,经IPTG诱导后收集菌体进行SDS-PAGE电泳分析.优化表达条件后纯化融合蛋白,用Western blotting鉴定其抗原性.结果表明,获得的HLS2 DNA片段与GenBank中的HLS2(序列号AB162827)序列的同源性为98%.构建的pET-30a-HLS2表达载体在大肠杆菌中表达了约为47 000的HLS2融合蛋白,主要以包涵体形式存在,IPTG终浓度为1.0 mmol/L、28℃诱导7 h后融合蛋白的表达量最高.Western blotting显示该融合蛋白可与兔抗长角血蜱阳性血清反应.     

Identification of genes encoding cement-like antigens expressed in the salivary glands of Haemaphysalis longicornis

A cDNA expression library from the salivary glands of hard tick, Haemaphysalis longicornis, was constructed. Immunoscreening was performed using sera of the rabbit repeatedly infested with ticks and seventeen positive clones were obtained. A BLASTP search suggested that 8 sequences matched with that of hypothetical H. longicornis sequence and one clone encoded HL35 antigen U from the same tick species. Eight of 17 gave no match to any sequence reported in the database. The proteins expected from these novel sequences possess common characteristics with cement proteins which assist ticks in their attachment to the host during blood feeding. The expression of these genes in salivary glands was confirmed by RT-PCR. Four of the 8 sequences showed to be upregulated upon blood feeding. These immunodominant antigens are of particular interest as candidates for future cement protein based-tick vaccine.