Characterization of Haemaphysalis longicornis recombinant cement-like antigens and preliminary study of their vaccination effects

Two genes encoding immunodominant antigens, hlim2 and hlim3, were obtained from a salivary gland cDNA library of the hard tick, Haemaphysalis longicornis. The recombinant proteins were expressed in Escherichia coli as the GST fusion protein and used for immunization. We observed that the attachment rate of nymphal ticks fed on mice immunized with GST-hlim3 was significantly lower than that in the control group during the initial days of feeding. However, immunization with GST-hlim3 did not affect the engorgement rate of the ticks. In sharp contrast, GST-hlim2 did not influence the attachment rate and feeding period of ticks but had a significant reduction in the engorgement body weight. These data highlight the suitability of the 2 recombinant cement-like proteins for use in a cocktail vaccine.     

Maturation of Theileria sergenti in salivary glands of Haemaphysalis longicornis induced by temperature stimulation

The parthenogenetic Haemaphysalis longicornis larvae engorged on cattle naturally infected with Theileria sergenti were reared at 24 degrees C. The resultant nymphal ticks were incubated at 37 degrees C to clear the effect of incubation on the development and maturation of sporozoites. The sporozoites in the salivary glands of the nymphal ticks exposed to 37 degrees C for 16 days were observed by the methyl green pyronin staining method. The ticks exposed to 37 degrees C were ground up in a mortar and the supernatant of the tick suspension in PBS was inoculated into cattle. The cattle showed parasitemia and specific antibody response 18 days after inoculation. Consequently, the parasites in the tick salivary glands became infective to cattle by incubating infected. H. longicornis nymphs at 37 degrees C.     

A novel gene encoding a thrombin inhibitory protein in a cDNA library from Haemaphysalis longicornis salivary gland

A novel thrombin inhibitory protein coding gene was identified from a cDNA library derived from salivary gland of partially-fed Haemaphysalis longicornis (hard tick). The gene encoded a 93-amino acid protein, designated chimadanin, which had a signal peptide sequence and was predicted to be a secretory protein. It showed no similarity to any other previously identified proteins or conserved domain sequences. The gene was expressed during blood feeding and suggested to be expressed mainly in the salivary gland. The predicted mature region of chimadanin was expressed in Escherichia coli and characteristics of the recombinant chimadanin were determined. The activated partial thromboplastin time and the prothrombin time in sheep plasma were significantly prolonged by chimadanin in a dose dependent manner. Amidolytic activity of thrombin was also inhibited by chimadanin in a dose dependent manner and it suggested that chimadanin was an anticoagulant with thrombin inhibitory activity. This newly identified thrombin inhibitor may play an important role in tick blood feeding.     

Identification of the tropomyosin (HL-Tm) in Haemaphysalis longicornis

Haemaphysalis longicornis tropomyosin (HL-Tm) was amplified by RT-PCR. The cDNA contained a 825 bp open reading frame coding for 274 amino acids with a predicted theoretical isoelectric point (pI) of 4.55 and molecular weight of 31.7 kDa. Real-time RT-PCR analysis showed that the expression levels of the HL-Tm in the unfed-females were significantly higher than in other tested developmental stages (eggs, unfed-larvae and unfed-nymphs). Western blot analysis showed that rabbit anti-serum against H. longicornis unfed-adult ticks recognized the recombinant HL-Tm protein (rHL-Tm). Immunization of rabbits with the rHL-Tm resulted in a statistically significant reduction of female engorgement and oviposition. Silencing of HL-Tm by RNAi showed a decrease in tick engorgement and oviposition, which is consistent with the effect of recombinant protein vaccine on the adults. These results showed that tick HL-Tm might be involved in the regulation of ticks blood-feeding, growth and oviposition.     

Characterization of proteolytic enzymes expressed in the midgut of Haemaphysalis longicornis.

The proteolytic activities present in midguts of both fed and unfed Haemaphysalis longicornis were assessed by using the gelatin-substrate gel electrophoresis and inhibitor sensitivity analyses. Three predominant (116, 48 and 48 kDa) and two weak (55 and 60 kDa) proteinase bands were commonly expressed in both unfed and fed ticks, while a weak 80 kDa band was only present in fed ticks. Consistent with observations on other tick species, proteolytic activity against the gelatin substrate was observed only under acidic conditions. Inhibition studies against the gelatin substrate using a panel of inhibitors showed that the predominant proteolytic enzymes of 40 and 48 kDa molecular mass are cysteine proteinases. These results are discussed in the context of host vaccination as an alternative tick control method to the current use of chemical acaricides.     

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

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.     

Identification of the heat shock protein 70 (HLHsp70) in Haemaphysalis longicornis

A Haemaphysalis longicornis heat shock protein 70 (HLHsp70) was identified from a cDNA library synthesized from tick eggs. The HLHsp70 cDNA is 2311 bp in length and encodes 661 amino acid residues with the predicted molecular weight of 72.5 kDa and an isoelectronic point (pI) of 5.2. It also contains the highly conserved functional motifs of the Hsp70 family and a specific endoplasmic reticulum (ER) retention signal "KDEL" that is common among ER-localized proteins. The HLHsp70 exhibits 90% amino acid identity to the putative Hsp70 of Ixodes scapularis, and 85% to Gallus gallus 78 kDa glucose-regulated protein precursor. Real time RT-PCR analysis showed that the expression levels of the Hsp70 in ovaries and salivary glands were significantly higher than in other tested tissues in partially fed females. Although the expression level of the HLHsp70 was constantly low in unfed ticks, it was significantly induced by blood-feeding. Further, the expression was positively correlated to the temperature (4-37°C, tested). Western blot analysis showed that the rabbit antiserum against the recombinant HLHsp70 protein (rHLHSP70) recognized bands of approximately 100, 72, and 28 kDa from egg lysates, as well as a 72kDa fragment in protein extracts from partially fed larvae. Immunization of rabbits with the rHLHSP70 did not result in a statistically significant reduction of female tick engorgement and oviposition. These results suggest that although HLHSP70 plays a role in the physiological activities of ticks, as a constitutive protein it was not suitable for selection as a candidate vaccine antigen against ticks.     

Random sequencing of cDNA library derived from partially-fed adult female Haemaphysalis longicornis salivary gland

A cDNA library was constructed from salivary glands of partially-fed adult female Haemaphysalis longicornis (hard tick). Randomly selected clones were sequenced and a total of 633 sequences were analyzed by bioinformatic programs. The sequences were grouped into 213 clusters, with each cluster being considered to be composed of mRNAs derived from the same gene or closely related genes. About 36% of the mRNA sequences showed significant similarity to known proteins in the non-redundant protein database by the NCBI blastx program and appeared to be coding for functional predicted proteins, whereas the remaining 64% had no similar sequences. Two thirds of the predicted proteins were annotated as basic cellular proteins (housekeeping proteins). Among the functional predicted protein sequences, other than the housekeeping proteins, several protease inhibitors including anticoagulants, two metalloproteases and a potential immunosuppressive protein could be identified. These proteins may play important roles during tick feeding and could be novel anti-tick vaccine candidates.     

双芽巴贝斯虫在长角血蜱肠内发育的形态学研究

以耳袋法将长角血蜱(Haemaphysalis longicornis)幼虫饲于实验感染双芽巴贝斯虫(Babesia bigemina)牛,幼虫饱血后24h内,其肠管内容物中红细胞内、外见有单梨子型(3.5～4.5μm×1.2～2.6μm)和双梨子型(4.1～4.8μm×1.8～3.0μm)两种裂殖体.饱血后24～48h,随着裂殖体细胞膜及核变性而发生形态变化.48～72h,绝大多数裂殖体出现溶解.72h后,这些裂殖体从肠道消失.其后,在蜱肠上皮及血淋巴中也未能找到双芽巴贝斯虫体.本实验从形态学上证明双芽巴贝斯虫在长角血蜱若虫肠道内不能发育.     

长角血蜱肠道细菌的分离与鉴定

为深入研究肠道细菌在蜱的生理学和传播病原上的作用,本研究进行了长角血蜱肠道细菌的分离鉴定。按照细菌传统分离方法并结合16S rDNA测序鉴定,获得7个分离株,这些菌株分别属于显核菌属(Caryophanon sp.)、考克氏菌属(Kocuria sp.)、葡萄球菌属(Staphylococcus sp.)、微杆菌属(Microbacterium sp.)和短杆菌属(Brevibacterium sp.)5个菌属。     

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.     

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.     

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.