Screening and Molecular Cloning of a Protective Antigen from the Midgut of Haemaphysalis longicornis

Vaccination is considered a promising alternative for controlling tick infestations. Haemaphysalis longicornis midgut proteins separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane were screened for protective value against bites. The western blot demonstrated the immunogenicity of 92 kDa protein (P92). The analysis of the P92 amino acid sequence by LC-MS/MS indicated that it was a H. longicornis paramyosin (Hl-Pmy). The full lenghth cDNA of Hl-Pmy was obtained by rapid amplification of cDNA ends (RACE) which consisted of 2,783 bp with a 161 bp 3'' untranslated region. Sequence alignment of tick paramyosin (Pmy) showed that Hl-Pmy shared a high level of conservation among ticks. Comparison with the protective epitope sequence of other invertebrate Pmy, it was calculated that the protective epitope of Hl-Pmy was a peptide (LEEAEGSSETVVEMNKKRDTE) named LEE, which was close to the N-terminal of Hl-Pmy protein. The secondary structure analysis suggested that LEE had non-helical segments within an α-helical structure. These results provide the basis for developing a vaccine against biting H. longicornis ticks.     

Ticks collected from selected mammalian hosts surveyed in the Republic of Korea during 2008-2009

A tick survey was conducted to determine the relative abundance and distribution of ticks associated with selected mammals in the Republic of Korea (ROK) during 2008-2009. A total of 918 ticks were collected from 76 mammals (6 families, 9 species) captured at 6 provinces and 3 Metropolitan Cities in ROK. Haemaphysalis longicornis (54.4%) was the most frequently collected tick, followed by Haemaphysalis flava (28.5%), Ixodes nipponensis (7.6%), Ixodes pomerantzevi (4.8%), Ixodes persulcatus (4.6%), and Haemaphysalis japonica (0.1%). Adults (57.0%) and nymphs (28.7%) of Ixodes and Haemaphysalis spp. were collected most frequently from medium or large mammals in this survey, while few larvae (14.3%) were collected. Hydropotes inermis was the most frequently captured mammal (52.6%), with a 16.4 tick index and 5 of 6 species of ticks collected during this survey. H. longicornis (69.7%) was the predominant tick collected from H. inermis, followed by H. flava (22.2%), I. persulcatus (6.1%), I. nipponensis (1.8%), and H. japonica (0.2%).     

Proteomic Screening of Antigenic Proteins from the Hard Tick,Haemaphysalis longicornis (Acari: Ixodidae)

Proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of proteome. For detection of antigens from Haemaphysalis longicornis, 1-dimensional electrophoresis (1-DE) quantitative immunoblotting technique combined with 2-dimensional electrophoresis (2-DE) immunoblotting was used for whole body proteins from unfed and partially fed female ticks. Reactivity bands and 2-DE immunoblotting were performed following 2-DE electrophoresis to identify protein spots. The proteome of the partially fed female had a larger number of lower molecular weight proteins than that of the unfed female tick. The total number of detected spots was 818 for unfed and 670 for partially fed female ticks. The 2-DE immunoblotting identified 10 antigenic spots from unfed females and 8 antigenic spots from partially fed females. Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF) of relevant spots identified calreticulin, putative secreted WC salivary protein, and a conserved hypothetical protein from the National Center for Biotechnology Information and Swiss Prot protein sequence databases. These findings indicate that most of the whole body components of these ticks are non-immunogenic. The data reported here will provide guidance in the identification of antigenic proteins to prevent infestation and diseases transmitted by H. longicornis.     

Comparative microbiome analysis of Haemaphysalis longicornis ticks at the Korea Combat Training Center in 2022

The Korea Combat Training Center (KCTC), located in Gangwon Province, is a restricted military training facility where research on the environmental conditions and health risks to military personnel has been limited. In this study, using iSeq 100, we investigated the bacterial abundance and microbiome of Haemaphysalis longicornis specimens collected at the KCTC from June to August 2022, to assess current and potential public health risks to military personnel. Our results show that adult ticks had significantly greater species richness compared with larvae and nymphs, with no notable differences in diversity across developmental stages. Principal coordinate analysis of the microbial communities did not show differences attributable to any single factor, such as collection location or date. Coxiella-like endosymbionts (AB001519) were identified in all 13 samples, and Jatrophihabitans, Sphingomonas, and Spirosoma were consistently found across all samples. In addition, iSeq 100 also identified Rickettsia rickettsii and Borrelia spp., which were not detected with conventional polymerase chain reaction (PCR).     

Cross-Sectional Survey of Ixodid Tick Species on Grazing Cattle in Japan

Ixodid tick species were collected from cattle in 60 grazing fields throughout Japan. Haemaphysalis longicornis was mainly recovered in the western and southern regions, while Ixodes species were collected mainly in the central to northern regions. Other tick species such as Amblyomma testudinarium, Boophilus microplus, H. flava and H. kitaokai were identified from a few fields in the central and southern regions. Haemaphysalis longicornis were recovered in the fields with higher temperatures and annual rainfall, whereas I. ovatus and I. persulcatus were collected in fields with lower temperatures and annual rainfall. Some of these tick species are capable of transmitting pathogens harmful to cattle and humans, so proper control strategies are required.     

长角血蜱卵黄蛋白单克隆抗体的制备及鉴定

为研究蜱类的卵黄发生及其机理,用长角血蜱Haemaphysalis longicornis卵黄蛋白（vitellin, Vn）免疫BALB/c小鼠,取免疫鼠脾细胞与骨髓瘤细胞SP2/0进行融合,经3次克隆化筛选,获得6株能稳定分泌抗Vn的单克隆抗体（McAb）,即1B5,2A7,2B8,2F2,3A1和3G1。1B5,2B8和2F2亚型为IgGA;2A7亚型为IgG1;3A1和3G1亚型为IgG2a。6株McAb均具有高度特异性,效价在1∶10.5以上。选取效价和特异性最好的1株抗体1B5进行SDS-PAGE分析和亲和力测定,测得1B5重链和轻链的分子量分别为58 kD和21 kD,亲和常数为2.8993×10^-6。Western免疫印迹分析发现6株单抗均与Vn的8个亚基发生免疫反应。本研究成功制备了6株抗长角血蜱Vn的单克隆抗体,为深入阐明长角血蜱卵黄发生的过程与调控机理提供了重要的工具。     

Goats,hares, and rabbits as hosts for the New Zealand cattle tick,Haemaphysalis longicornis

Abstract

Feral goats and hares were commonly infested by immature stages of the New Zealand cattle tick, Haemaphysalis longicornis. No explanation could be found for the low prevalence of adult ticks on these hosts. The ears of both host species were almost the exclusive feeding site of the ticks and this may be a consequence of grooming behaviour. Another potential host, the rabbit, was examined but few were found to be infested.

The less restricted range of non-domesticated hosts, together with feeding habits that differ from domestic stock, make them an important additional source of information on the ecology and seasonal pattern of activity of H. longicornis. Also, they are a source of contamination for tick-free pasture, and could possibly maintain the tick population in the absence of sheep and cattle. It is important that their role as alternative hosts be understood and considered in tick-control programmes.     

Immunization effect of recombinant P27/30 protein expressed in Escherichia coli against the hard tick Haemaphysalis longicornis (Acari: Ixodidae) in rabbits

We investigated the induction of resistance to Haemaphysalis longicornis infestation in rabbits that had been immunized with recombinant H. longicornis P27/30 protein. The success of immunological control methods is dependent upon the use of potential key antigens as tick vaccine candidates. Previously, we cloned a gene encoding 27 kDa and 30 kDa proteins (P27/30) of H. longicornis, and identified P27/30 as a troponin I-like protein. In this study, rabbits that were immunized with recombinant P27/30 expressed in Escherichia coli showed the statistically significant longer feeding duration for larval and adult ticks (P<0.05), low engorgement rates in larval ticks (64.4%), and an apparent reduction in egg weights, which suggest that H. longicornis P27/30 protein is a potential candidate antigen for a tick vaccine. These results demonstrated that the recombinant P27/30 protein might be a useful vaccine candidate antigen for biological control of H. longicornis.     

实验室条件下长角血蜱的生物学特性研究

实验室条件[(27±1)℃;75%RH;6L：18D]下,长角血蜱完成其生活史需135.8d：卵的孵化期为38.5d：幼虫吸血前期、吸血期和蜕皮前期分别为5.3d,3.8d和13.9d;若虫吸血前期、吸血期和蜕皮前期分别为7.2d,5.4d和16.9d;成虫吸血前期、吸血期、产卵前期和产卵期分别为7.6d,9.4d,7.8d和20.0d。雌虫饱食体重与产卵量之间存在非常显著的正相关(r＝0.9496,P<0.001)。雌虫的生殖效率指数REI＝11.06,生殖适合度指 数RFI＝7.19。生活周期在不同季节无明显变化。雄虫吸血期受雌虫吸引而成功地交配,这一过程是在吸引性信息素的作用下完成的。     

性信息素2，6-二氯酚在长角血蜱交配行为中的作用

长角血蜱Haemaphysalis longicornis的交配行为包括7个时期,行为的完成依赖于性信息素的调节。生物测定表明：雄蜱的行为反应受雌蜱分泌的性信息素影响。堵塞雌蜱盾窝其行为受到抑制,点滴2,6-DCP或雌蜱盾窝腺提取物则被恢复。用气相色谱法测定了雌蜱盾窝腺中2,6-DCP的含量;吸血后1～２天含量最高(11.12 ng/只);吸血后3～５天即交配前下降交维持在一较恒定的水平;吸血后6～７天即交配后明显降低;饱血后检测不到2,6-DCP。2,6-DCP是长角血蜱性信息素的一种成分。