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.     

A recombinant Eimeria protein inducing interferon-gamma production: comparison of different gene expression systems and immunization strategies for vaccination against coccidiosis

A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.     

Molecular Cloning and Expression of IL2 cDNA from the Tibet Pig

Interleukin-2 is a vital cytokine secreted by activated T lymphocytes, and plays important role in the regulation of cellular and humoral immunity of animals. In our experiment, IL2 cDNA of the Tibet Pig was first cloned by RT-PCR from ConA-stimulated lymphocytes in the blood and subcloned into pMD-18 T vector, which then was identified with endonuclease restriction. The sequencing result showed that Tibet pig IL-2 (TPIL-2) cDNA was 503 bp long (ORF was 465 bp) (Genbank accession number: AY 294018). The recombinant prokaryotic and eukaryotic expression plasmids of the cDNA were then constructed to analyse the ability to stimulate the proliferation of porcine lymphocytes in vitro. The recombinant porcine IL-2 expressed in the prokaryotic cells was found to be of 43 kDa molecular mass, which was consistent with a 17.4 kDa protein deduced from the IL-2 cDNA sequence (glutathione S-transferase molecular mass is 26 kDa); the recombinant protein in eukaryotic cells was confirmed by use of specific rabbit anti-porcine IL-2 serum in an ELISA. The bioactivity of TPIL-2 was detected through MTT colorimetry by stimulating the proliferation of pig ConA-stimulated blasts in vitro. The results indicate that the TPIL-2 significantly promoted the proliferation of ConA-stimulated blasts of pig. This confirms that IL-2 cDNA of the Tibet pig was successfully cloned and expressed in prokaryotic and eukaryotic cells, which lays the foundation for the the preparation of specific recombinant IL-2 protein and development of novel immune adjuvants to raise the immunity of pigs against various infectious pathogens and increase the immunoprotective efficacy of vaccines.     

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.     

亚洲璃眼蜱唾液腺多肽Ha-11的鉴定、表达和生物学功能的初步研究

本研究对亚洲璃眼蜱多肽Ha-11的编码基因进行了克隆、表达和初步的功能分析。该基因开放阅读框（open reading frame,ORF）为372 bp,编码123个氨基酸,其中含23个氨基酸的信号肽。将去除信号肽的编码区基因亚克隆至pGEX-4T-1载体,大肠杆菌中诱导表达,获得了37 kDa重组蛋白。抗重组Ha-11蛋白的免疫血清识别半饱血雌蜱的唾液腺中约11 kDa的天然蛋白。Real-time PCR结果表明该基因在亚洲璃眼蜱的各发育阶段、各组织中均有表达,但以若蜱表达量最高,而组织中以淋巴液表达量最高。重组蛋白在体外可以抑制脾细胞增殖和细胞因子IL-2、IFN-γ的分泌。研究结果显示Ha-11在蜱吸血过程中可能具有抗炎作用。     

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.     

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.     

Control of Boophilus microplus ticks in cattle calves by immunization with a recombinant Bm86 glucoprotein antigen preparation.

Forty Egyptian native cattle calves of 4-6 months old randomly allocated into two groups of twenty animals each were used to assess the effect of immunization of animals with a recombinant Bm86 antigen derived from Boophilus microplus ticks on induction of immunity that could protect calves during tick season. The immunization protocol involved two injections administered intramuscularly, the first was applied with complete Freund's adjuvant and the second was given with incomplete Freund's adjuvant two months later. Control calves were given saline plus adjuvant. Immunization reduced the number of adult ticks developing from a subsequent challenge infestation by 78% in immunized calves. Vaccination also, significantly reduced the weight of adult ticks in immunized calves (30.51%). The results of skin delayed hypersensitivity reaction revealed that the diameter of sites injected with the recombinant Bm86 antigen was significantly larger in immunized calves than those in controls. Analysis of the immune response indicated that there was a significant increase in the level of IgG and IgA antibodies in serum of immunized calves and protection from reinfestation was correlated with the levels of circulating antibodies.     

狂犬病病毒SRV9克隆株核蛋白基因的克隆、表达与特性分析

根据已发表的狂犬病病毒核蛋白基因序列，设计并合成了一对引物，从SAD株驯化的SRV9。蚀斑株中提取病毒RNA，通过RT-PCR扩增出核蛋白的全长cDNA序列，测序结果显示，其序列与国外报道的SAD母源株序列一致。将核蛋白的cDNA克隆至原核表达载体pET-28b( )中，转化大肠杆菌BL21（DE3）plyss，于30℃1mmol/LIPTG条件下诱导表达，大肠杆菌菌体裂解产物经SDS-PAGE分析，在分子量约为56kDa处出现一新的蛋白带。和预期的目的蛋白分子量相符，Western-blotting检测表明，表达产物能与狂犬病病毒阳性血清发生特异性反应，出现单一反应带，扫描分析显示，表达产物占菌体总蛋白的23%，包涵体分离，纯化后，纯度达89%，上述结果为核蛋白在狂犬病基因免疫和免疫检测中的进一步应用奠定了基础。     

Efficacy of a single whole-body spray treatment of spinosad, against Boophilus microplus (Acari: Ixodidae) on cattle.

The efficacy of a single whole-body spray of spinosad, a naturally derived control agent, applied at three concentrations was evaluated against cattle infested three separate times prior to treatment and at four weekly intervals following treatment with Boophilus microplus (Canestrini). At 0.0167% active ingredient (AI) both tick numbers (1894 ticks per calf) and index of fecundity (IF) of females (258.3) were no different than that of the control group. However, spinosad treatment at both 0.05 and 0.15% AI resulted in fewer ticks per calf (600 and 935, respectively) with lower IF values for females (43.4 and 38.4, respectively). The percent control of ticks on the animals at the time of treatment (acute efficacy) was dramatically lower at 0.0167% AI (21.4%) than at 0.05 (86.3%) and 0.15% AI (87.9%). Spinosad treatments appeared to be more effective against immature stages (nymphs and larvae) than against adult ticks that were on the animals at the time of treatment. The mean weight of females that survived to repletion was similar (322-348 mg) in all groups. By contrast, the mean weight of egg masses produced by females was highest in the control group (155 mg), whereas each increase in spinosad concentration resulted in a substantial decrease in egg mass weight, with the 0.15% AI group averaging only 73 mg. The hatch rate of eggs derived from females ranged from 93.4% in control females down to 53.9% hatch for females treated at 0.15% AI spinosad. The residual efficacy of spinosad at 0.0167% AI was poor even at 1 week following treatment, resulting in 101 ticks per calf and a level of control of only 66.4%. At 0.05% AI, protection against successful reinfestation was high at 1-week post-treatment where only five ticks per calf reached repletion, and control of the IF of these females was 99.3%. The 0.15% AI treatment provided almost complete protection against reinfestation for 2 weeks following treatment (< or =5 ticks per calf), and control of the IF of these ticks was >99.9%. Thus, the use of spinosad at US ports-of-entry would be unacceptable because of the critical necessity of achieving 100% control with a single treatment to prevent the reintroduction of ticks. However, it is likely ticks could be eradicated using spinosad in tick infested areas of the US if repeated (systematic) treatments were applied to cattle maintained on the premises.     

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 µg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.     

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.     

亚洲璃眼蜱唾液腺差异表达基因文库的构建及分析

从单雌蜱克隆群中挑选未吸血雌蜱60只，随机分成两组。未吸血组直接剖取唾液腺．半饱血组于蜱吸血第5d采集，分离唾液腺。Trizol法提取总RNA，经第一链合成、LD—PCR、RasⅠ酶切、接头连接和抑制消减杂交（SSH）等步骤，获得差异表达基因的cDNA片段。将纯化的cDNA片段与pGEMT—Easv我体连接，转化DH5a，获得204个白色菌落。扩增检查表明，136个克隆含有插入片段，片段大小为250bp-850bm，测出有效序列120个。由10个cDNA片段的RT—PCR检查结果初步断定，本研究消减效果良好。由网上资源分析得：21个片段与其他蜱的基因，19个片段与按蚊、库蚊、钩虫、奥斯特线虫等其他吸血寄生虫的基因，9个与果蝇的基因具有同源性。     

Efficacy of a combination of imidacloprid 10%/permethrin 50% versus fipronil 10%/(S)-methoprene 12%, against ticks in naturally infected dogs

Preventing tick bites is a fundamental step towards reducing the impact of tick-borne protozoal, bacterial and viral diseases (TBDs) in humans and animals. The aim of this study was to evaluate the efficacy of a combination of imidacloprid 10%/permethrin 50% and of fipronil 10%/S-methoprene 12% against ticks in naturally infected dogs and to assess methodological parameters to calculate drug efficacy on tick immature stages.

From July to August 2004, 45 privately owned dogs of various sexes, ages, breeds, coat length and habits were enrolled in a trial carried out in an area (radius approximately 50 km) in Southern Italy. Three homogeneous groups (both for dog population and tick population) were formed: 15 dogs treated with imidacloprid 10% and permethrin 50% spot-on (group A), 15 dogs treated with fipronil 10% and methoprene 12% spot-on (group B) and 15 untreated dogs (group C). The dogs in each group were then sub-grouped according to their age and weight. Two different treatments were administered (time 0 and +28 days) to groups A and B, and the dogs were checked weekly for tick infestation until day +56 post-treatment (p.t.). Twenty-four areas distributed on the whole body surface were examined for ticks at each follow-up, while only at time 0 and at day +56 p.t., ticks were collected from the dogs and identified.

For the immature stages a semi-quantitative method was adopted and the load of immature stages was evaluated and grouped into four classes up to day +56 p.t. when the mean number of immature ticks (MIT) for each infection class was evaluated.

All the adult ticks collected were identified as brown dog ticks (Rhipicephalus sanguineus). Immature stages were first compared at day +28 p.t.. The efficacy of both products used in groups A and B on adult ticks was high and generally very similar. Conversely, the efficacy of imidacloprid 10% and permethrin 50% against immatures was higher than that of fipronil 10% and methoprene 12% throughout the observation period with statistically significant differences (p < 0.05) at day +28 p.t. (i.e. group A = 98.52%, group B = 72.40%).

On the whole, in analysing the efficacy of both products against adult plus immature ticks, it was found that the combination of imidacloprid 10% and permethrin 50% was more effective than fipronil 10% and methoprene 12%, with the differences being statistically significant at day +28 p.t. (group A = 98.43%, group B = 77.56%).