不同饲喂方式和温度对长角血蜱甘肃株生物学特性的影响

在不同饲喂方式和温度条件下,观察我国北方长角血蜱(Haemaphysalis longicornis)甘肃株的生活史及对其生物学特性的影响。将试验蜱分组后置于不同饲喂方式和温度下,待其发育到下一阶段,叮咬实验动物,连续调查其发育阶段的生物学特性。研究发现,完成一个世代需101 d～148 d。成年饥饿雌蜱吸血期具有两个显著特征:缓慢吸血和快速吸血;经兔体叮咬的成年雌蜱其饱血体重小于羊体。不同饲喂条件下长角血蜱饱血体重、产卵量差异极显著(P0.01);在一定温度范围内,幼蜱、若蜱的蜕变时间随温度的升高而缩短;当饱血雌蜱处于发育阶段时,产卵前期、产卵期及卵的孵化期均随着温度的升高而缩短;16℃～30℃条件下长角血蜱产卵量没有变化,利于蜱的存活;4℃以下和45℃以上饱血蜱及其后代不能存活;16℃以下蜱的发育周期延长。结果表明,长角血蜱为三宿主蜱;雄蜱可以帮助雌蜱吸血;在羊体上的吸血能力优于在兔体上的吸血能力;温度对长角血蜱的发育繁殖有影响。     

长角血蜱不同龄期虫体抗原分析

用琼脂双向扩散法、酶联免疫吸附技术和蛋白质免疫印迹技术，用长角血蜱三个龄期的虫体蛋白检测其幼虫、若虫和成虫未吸血全蜱匀浆液分别免疫兔后的血清效价。结果表明不同生活阶段的虫体之间存在免疫交叉反应。实验结果表明不仅长角血蜱三个龄期虫体之间存在共同抗原，而且与微小牛蜱幼虫和越南血蜱幼虫之间也存在共同抗原。     

长角血蜱不同龄期虫体抗原分析

用琼脂双向扩散法、酶联免疫吸附技术和蛋白质免疫印迹技术,用长角血蜱三个龄期的虫体蛋白检测其幼虫、若虫和成虫未吸血全蜱匀浆液分别免疫兔后的血清效价。结果表明不同生活阶段的虫体之间存在免疫交叉反应。实验结果表明不仅长角血蜱三个龄期虫体之间存在共同抗原,而且与微小牛蜱幼虫和越南血蜱幼虫之间也存在共同抗原。     

卵形巴贝西虫在长角血蝉若蜱唾液腺内的发育

本实验以感染有卵形巴贝西虫的长角血蜱若蜱叮咬家兔,对若蜱从叮咬到饱血脱落为止5d内唾液腺中卵形巴贝西虫的发育进行了观察.结果证实,卵形巴贝西虫在若蜱唾液腺细胞内经过母孢子(Sporont)阶段发育为子孢子(Sporozoite),因此卵形巴贝西虫取孢子生殖方式.随着子孢子的增加与释放,唾液腺细胞出现明显的空洞化.     

半散放麋鹿长角血蜱病的诊治

长角血蜱（Haemaphysalis Longicorni,）是属蛛形纲、蜱螨目、硬蜱科、血蜱属的一种节肢动物。分布较广泛，未成熟吸血时体小而不易引人注意。由于生活方式的多样性，它寄生于多种动物体表，对人也有侵袭，并且许多性疾病的传播媒介，对此国内早有研究。但长角血蜱寄生半散放麋鹿体表而致病死亡的情况尚未有报导，     

长角血蜱保护性抗原基因P27/30的克隆和原核表达

采用RT—PCR技术首次从孤雌生殖长角血蜱四川株克隆到P27／30基因,扩增序列全长670bp,包含完整的开放阅读框,编码201个氨基酸,预测蛋白相对分子质量为23．38ku。同源性分析表明孤雌生殖长角血蜱中国株与日本株P27／30基因同源性高达99．85％。经RT—PCR检测分析,该基因在孤雌生殖长角血蜱的卵、幼蜱、若蜱、饥饿成蜱和饱血成蜱这几个阶段均有表达。将该基因亚克隆后连接到pET32a（＋）原核表达载体,转化BL21（DEa）宿主菌,经IPTG诱导可成功进行表达。表达的目的蛋白大小为24ku左右,与预期大小一致;Western-blot显示兔抗长角血蜱全虫抗体能够识别该重组表达蛋白。     

哒螨灵和高效氯氰菊酯对长角血蜱饱血雌蜱的毒力对比

为丰富灭蜱药物种类,获得对长角血蜱有杀灭效果的新药,本试验将哒螨灵、高效氯氰菊酯均设成2mg/kg、10mg/kg、50mg/kg、250mg/kg、1250mg/kg、6250mg/kg 6个浓度对长角血蜱饱血雌蜱进行体外浸渍法处理,观察蜱的死亡和产卵情况,并经数据处理得到两种药物对蜱的半数致死量、产卵指数(IE)、产卵抑制率等指标及统计图,然后通过多指标评价对比哒螨灵和高效氯氰菊酯的灭蜱效果。结果显示,哒螨灵的半数致死量小于高效氯氰菊酯,说明哒螨灵对饱血成蜱的毒力比氯氰菊酯强;在药物浓度大于50mg/kg时,两种药的产卵抑制率很接近。结果表明,在杀伤长角血蜱的饱血雌蜱作用中,哒螨灵可以达到和氯氰菊酯同样的效果。哒螨灵高效、低毒、低残留,可将其作为灭蜱新复方制剂成分进行研究开发。     

镰形扇头蜱重组肌钙蛋白I抗蜱免疫保护效果的初步分析

为了评估镰形扇头蜱肌钙蛋白I（TroponinI,TnI）作为候选疫苗分子的潜力,应用肌钙蛋白I重组蛋白进行了抗蜱免疫实验。将重组蛋白按常规方法免疫新西兰兔,三次免疫后分别用若蜱、成蜱进行攻虫实验,观察其免疫保护效果。结果发现蜱的吸血行为受到一定的影响,与对照相比,若蜱和成蜱吸附率、饱血率、饱血体重和死亡率有显著差异,而在饱血时间上差异不显著,说明TnI基因的重组表达产物具有一定的免疫保护作用。     

长角血蜱的免疫研究进展

以长角血蜱疫苗候选抗原为例，从长角血蜱蛋白水解酶、蜱生殖和发育相关分子及RNA干扰技术、IgG结合蛋白技术几方面概述了近年来国内外有关蜱疫苗的研究现状，并展望了蜱疫苗的发展趋势。     

卵形巴贝斯虫在长角血蜱幼虫体内的发育观察

文献报道,巴贝斯虫可经卵传递。然而有关经卵传递时巴贝斯虫的形态学研究尚未见报道。本实验以长角血蜱成虫对卵形巴贝斯虫感染牛饱血脱落后的次代幼虫叮咬除脾实验牛,对幼蜱体内卵形巴贝斯虫的发育进行形态学观察,同时幼蜱叮咬后实验牛末梢血液进行观察研究。     

Comparison of macrophage scavenger receptor-A knockout mice with wild type ones in the immune response against repeated infestation with Haemaphysalis longicornis

Using macrophage scavenger receptor-A knockout (SRKO) mice, we examined the role of macrophage class A scavenger receptors (MRS-A) on the immune response and acquisition of host resistance against repeated infestation with Haemaphysalis longicornis. Except for one batch of nymphs that infested one of the SRKO (SR-/-) mice and showed no appreciable reduction in body weight, all the other groups of nymphs manifested significant decrease in body weight. Both SR-/- and wild type (SR+/+) mice showed a sustained increase in anti-tick antibody titers, but SR+/+ mice showed significantly higher titers. The IFN-gamma assayed in SR-/- mouse immune sera was substantially less compared with that in SR+/+ mice. Immune sera from SR-/- and SR+/+ mice recognized the 51 and 44 kDa, and 44 kDa proteins, respectively, of the salivary gland antigen. The difference in the level of anti-tick resistance manifested by both groups of mice may be influenced by less efficient trapping and processing of tick antigens by macrophages in mice lacking for the macrophage scavenger receptors, and consequently affected the cascade of Th1 and Th2 responses. We have thus obtained valuable data that strongly infer the role of MSR-A in enhancing host defense against repeated infestation with H. longicornis.     

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.     

Relationship between the resistance of cattle to Haemaphysalis longicornis and to Boophilus microplus

Cattle that had been exposed to Haemaphysalis longicornis were as susceptible to Boophilus microplus as cattle that had never been exposed to either species of tick. Cattle with acquired resistance to both species ranked consistently for levels of resistance to each when infested separately. Concurrent infestation with H. longicornis had no effect on ranking for resistance to B. microplus. The coefficient of concordance between the rankings of individuals on their levels of resistance to both species of tick was positive, but was not statistically significant. We conclude that the tick antigens that stimulate host resistance are species-specific and do not cross protect. The apparent correlation in rankings for resistance to the 2 species may be a consequence of either an individual's immunological responsiveness to tick antigens or to non-specific host factors which determine levels of resistance. The apparent correlation suggests that co-selection for resistance to different tick species is practicable.     

Infection rates of Theileria sergenti in Haemaphysalis longicornis ticks collected from the field in Japan

For a period of one year Haemaphysalis longicornis ticks were collected in a pasture from vegetation by dragging flannel cloth and from soil samples at monthly intervals. Nymphal ticks were assessed for Theileria infection. Salivary glands were stained with methylgreen-pyronin and examined for parasite masses. Nymphal H. longicornis infected with Theileria sergenti were found in all samples during 12 months including the winter. After shortening the prefed period on rabbits to 24 hours, the parasite masses could be detected in the salivary glands of nymphs collected in the grazing season, from May to October, while no parasite masses were detected in other season. It was suggested that the environmental factors in the grazing season might influence on the maturation of parasites in the salivary glands of ticks.     

Morphological, biological and molecular characteristics of bisexual and parthenogenetic Haemaphysalis longicornis

The reproductive mechanism of Haemaphysalis longicornis is quite different from many other animal species. In this article, several characteristics of parthenogenetic and bisexual populations of H. longicornis were analyzed, including some important micro-structures, synchronized life cycle feature and sequences of mitochondrial 16S rRNA gene. The results suggested even though many observations of the two populations were similar to each other, some important differences also existed. The genital apron of parthenogenetic females was wider than that of bisexual females. Parthenogenetic individuals (except engorged females) were significantly larger in weight than bisexual individuals (p<0.01; unfed nymph p<0.05); the difference of nymphal premoulting, female feeding and preoviposition, and egg incubation periods between the two populations were significant (p<0.01); hatch percentage of parthenogenesis was lower than that of bisexual population (69% and 73%, respectively); parthenogenetic individuals had a slightly longer development cycle than bisexual individuals (134 and 129days, respectively). Hybridization attempts failed between them. Comparing to the two strains of bisexual H. longicornis, parthenogenetic strain of H. longicornis inserted two nucleotides of thymine, though the genetic distance of 16S rDNA between the bisexual and parthenogenetic populations was 0. In order to clarify the relationships of the two reproductive populations of H. longicornis, the sequences of 16S rDNA of four strains of two other species were also analyzed. The divergence between Hebei and Xinjiang strains of Rhipicephalus sanguineus was 0, and the sequences were totally identical between them. The divergence between the two subspecies of Hyalomma asiaticum was 0.01. The results indicated that the relationship between bisexual and parthenogenetic H. longicornis was certainly closer than that between subspecies, but farther than that between the same reproductive populations of conspecies.     

Influence of sex on the resistance of sheep lambs to an experimental Haemonchus contortus infection

36 intact male and 69 female lambs of two breeds (n = 63 Merinoland; n = 42 Rh?n) aged 12 weeks were orally infected with 5000 infective-stage larvae, L3, of the nematode Haemonchus contortus. After 4 and 8 weeks faecal egg counts (FEC), haematocrit values and plasma testosterone levels were determined. All lambs were slaughtered at 20 weeks of age. The gastrointestinal tracts were examined for the presence of adult stages of H. contortus. Male lambs showed significantly higher log FEC (p < 0.001), higher mean establishment rates (p < 0.05), higher worm burdens (p < 0.01) and lower haematocrit values (p < 0.001) when compared with female lambs. Correlations between economically important traits (body weight, daily weight gain) and parasitological parameters were significantly higher in male animals. Testosterone level was 4 weeks after infection significantly positive correlated with worm burden. The results suggest that female lambs are more resistant against an experimental H. contortus infection when compared with male lambs. Testosterone seems to play an important role in resistance. This approach can be of importance if parasite resistance is incorporated into breeding programs and the estimated breeding values for rams are only based on male offspring information. Therefore male breeding values are probably not representative for the whole population.     

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.     

Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901).

Molecular evidence that suggests the possible role of the ixodid tick, Haemaphysalis longicornis and its eggs in the transmission of equine Babesia caballi parasites is presented herein. Using polymerase chain reaction (PCR) to assay for DNA in parasites, presumably acquired by ticks that were allowed to feed on splenectomized-SCID mice, experimentally exposed to in vitro-cultivated B. caballi, we have obtained positive bands that corresponded to the expected B. caballi-specific 430bp gene fragment in 50% of female ticks used, and in 75 and 25% of eggs and larval progeny, respectively. Also, parasite DNA was detected in ticks, eggs and larvae as late as the 16th to the 20th day post-host infestation. Present findings support to the potential role of H. longicornis in the transmission of B. caballi parasites. Its capability, however, to successfully transmit the infection to horses under natural conditions in the field needs to be further ascertained. To our knowledge, this is the first documented study incriminating H. longicornis as a most and likely biological vector of equine babesias.     

Post natal ontogeny of immunological responsiveness in Merino sheep

Sheep in five age groups (two weeks, 10 weeks, 18 weeks, six months and four years old) were immunised systemically, twice, with ovalbumin or Brucella abortus (live or killed) and antibody responses in blood were measured. The animals were also infected with the nematode parasites Trichostrongylus colubriformis and Haemonchus contortus and faecal egg counts and serum antibody responses to larval antigens were measured. The experiments were designed so that, as far as possible, the effect of age per se could be dissociated from the combined effects of age and prior exposure to antigen. The effects of the age of sheep were more marked for antibody responses to Brucella abortus lipopolysaccharide than to ovalbumin. Older animals had much greater resistance to infection with internal parasites, as shown by the magnitude of the faecal egg count. In contrast to older lambs, neonatal lambs (infected with H contortus at two weeks old) had consistently declining concentrations of anti-H contortus antibody in their serum, mounted no detectable autogenous anti-H contortus antibody response in blood and appeared to develop no resistance to the parasite. Post natal ontogeny of immune responses was different for the various antigens/pathogens.