Haemaphysalis longicornis calreticulin is not an effective molecular tool for tick bite diagnosis and disruption of tick infestations.

Tick calreticulin (CRT) is a calcium-binding protein secreted into the host during blood feeding. It has been used as a biomarker of tick exposure and has potential as an anti-tick vaccine, but there is no information about these uses for Haemaphysalis longicornis CRT (HlCRT). We synthesized recombinant H. longicornis CRT (rHlCRT) and evaluated its potential for tick bite diagnosis and for disrupting tick infestations. The responses of mice and rabbits exposed to H. longicornis ticks were measured with ELISA to determine the antibody level against rHlCRT. To evaluate the effects of rHlCRT-induced anti-tick immunity, engorgement weight, tick engorgement index (TEI), feeding duration, ecdysis rate, and egg weight per engorged tick were compared between ticks fed on immunized and normal mice. Mean anti-tick CRT antibody levels in sera collected from mice at 1 and 15 days after primary tick exposure were not significantly different from the mean antibody levels in negative control mice that were not bitten by ticks (all P values > 0.05). No significant anti-HlCRT IgG responses developed in mice after second exposure to tick bites compared with the level of anti-HlCRT antibody response in negative control mice (all P values > 0.25). For rabbits, no significant differences in the antibody levels were observed in animals before challenge infestation and after tick exposures, and in animals after two tick exposures (all P values > 0.10). There were no significant differences in the body weight of ticks fed on immunized and normal mice (all P values > 0.15). No significant differences in TEI were observed between ticks fed on immunized mice and normal control mice (all P values > 0.50). There were no significant differences in feeding duration for female ticks, and feeding duration and ecdysis rate for nymphs in the experimental and control groups (all P values > 0.10 for feeding duration and P value = 0.19 for ecdysis rate). We did not observe a significant difference in egg weight per tick in the rHlCRT-immunized and the control groups (P = 0.88). HlCRT in H. longicornis tick saliva proteins appears to be nonimmunogenic to mammalian hosts like mice and rabbits. Vaccination with rHlCRT did not generate effective immunity against parthenogenetic and bisexual H. longicornis nymphs or female ticks. These results indicate that HlCRT is not a suitable molecular candidate for H. longicornis tick bite diagnosis and not effective for the disruption of tick infestations.

Zheng W ，Hu H ，Jiang J ，Sun X ，Fu R ，Tao H ，Liu Y ，Chen H ，Ma H ，Chen S ... -  《-》

Subolesin vaccination inhibits blood feeding and reproduction of Haemaphysalis longicornis in rabbits.

Lee SH ，Li J ，Moumouni PFA ，Okado K ，Zheng W ，Liu M ，Ji S ，Kim S ，Umemiya-Shirafuji R ，Xuan X ... -  《Parasites & Vectors》

Hemalin vaccination modulates the host immune response and reproductive cycle of Haemaphysalis longicornis.

Haemaphysalis longicornis can transmit high varieties of tick-borne pathogens (TBPs), and a primary strategy for preventing the transmission of those TBPs is to control ticks. Hemalin, a thrombin inhibitor of the Kunitz-type family and a crucial component in H. longicornis feeding process has been isolated from parthenogentic ticks. This study aimed to evaluate the validity of a recombinant Hemalin (rHlHemalin) vaccination as an anti-tick vaccine against H. longicornis in rabbits to find a new candidate for an effective tick control. In this study, mouse splenocytes were isolated and used to investigate immune responses after rHlHemalin stimulation. The rabbits were vaccinated with the rHlHemalin protein. After tick challenges, body weight at engorgement, egg mass, and the reproductive cycle of H. longicornis were evaluated. To confirm the vaccination, the passive immunization tests of α-rHlHemalin sera were performed. The results showed that the rHlHemalin protein could stimulate cytokine production in mouse splenocytes. Vaccination assay revealed that the periods from tick infestations to egg-hatch in the vaccination group were significantly longer than those in the phosphate buffer saline (PBS) group (P = 0.0003). In addition, the tick body weight at engorgement (P = 0.0019) and egg mass at 10 days after oviposition (P = 0.0232) were higher than those in the PBS group. These findings were consistent with the current passive immunization results and suggest rHlHemalin vaccination extended the reproductive cycle in H. longicornis but did not decrease the body weight at engorgement or weight of egg mass. Therefore, it is debatable whether Hemalin vaccination is highly-effective anti-tick vaccine or not. However, due to the importance of thrombin inhibitors in tick blood feeding and blood digestion, additional inhibitor-based vaccines should be developed aiming to find an effective and environmentally friendly biological strategy to combat ticks.

Ma H ，Ai J ，La Y ，Zhao X ，Zeng A ，Qin Q ，Feng S ，Kang M ，Sun Y ，Li J ... -  《-》

Comprehensive analysis of protein expression levels and phosphorylation levels in host skin in response to tick (Haemaphysalis longicornis) bite.

Ticks are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens. The blood sucking of ticks causes varying degrees of damage to the skin of the host. Proteins related to immune regulation, vascular repair, and wound healing in mammalian skin respond to tick bites by regulating their expression and post-translational modifications to protect the skin from injury. Phosphorylation of proteins, as the most common post-translational modification of proteins, plays an important role in the rapid regulation of cell signal transduction, gene expression and cell cycle. To systematically explore the molecular regulatory mechanisms employed by mammalian skin to resist tick bites, larval, nymphal, and adult Haemaphysalis longicornis were used to bite the skin tissues of healthy rabbits in the present study. The quantitative proteomic technology data-independent acquisition was then carried out to investigate in depth the changes in protein expression and phosphorylation in rabbit skin after tick bite. The results showed that among the 4034 proteins and 1795 phosphorylated proteins identified, a total of 202 proteins and 435 phosphorylation sites were changed after H. longicornis bite. In order to provide convenience for sucking blood, active substances in the saliva of H. longicornis injected into the rabbit's skin can cause the expression level of trichohyalin and peptidyl arginine deiminase 3 in the skin of the host downregulate, which can make the host hair loss and regeneration disorders. At the same time, the active substances in saliva of the H. longicornis led to the phosphorylation of microtubule actin cross-linking factor 1 in the host's skin and further inactivation, so as to delay the healing of the host wound. In response to tick bites, the host skin promotes coagulation through high expression of fibrinogen and fibronectin, and vascular repair through high expression of integrin linked kinase and tenascin C, as well as accelerated phosphorylation of the phosphorylated protein Nck adaptor protein 1, and wound healing through high expression of ezrin and integrin. The upregulation of proteins such as coronin, NADPH oxidase, calnexin, and calreticulin and phosphorylation level of IL-4R in the host skin after the H. longicornis bite indicated that the immune response was playing an important defensive role in response to tick bites. Meanwhile, we found that the upregulated two lectins, mannose receptor C-type 1 and DC-SIGN, may serve as molecular makers to identify and monitor whether the skin is bitten by ticks. SIGNIFICANCE: Haemaphysalis longicornis are parasitic arthropods that suck blood from the surface of most vertebrates. They can transmit a variety of pathogens and are harmful to humans and livestock. The present study is the first quantitative proteomic study on protein expression levels in the rabbit skin after infection by H. longicornis. It is also the first quantitative phosphoproteomic study in the host skin infected by ticks. In this study, we found that tick bites cause the host hair loss and regeneration disorders. For resisting tick bite, the host activates the immune response and initiates vascular repair and wound-healing systems. In addition, some phosphorylated proteins promote host immunity and vascular repair. These results can help us further understand the defence mechanism of the host against tick bites, provide a basis for the development of an anti-tick vaccine, the development of anti-tick drugs, and the diagnosis of tick-borne diseases.

Zhang X ，Zhang B ，Masoudi A ，Wang X ，Xue X ，Li M ，Xiao Q ，Wang M ，Liu J ，Wang H ... -  《-》

Efficacy of recombinant enolase as a candidate vaccine against Haemaphysalis longicornis tick infestation in mice.

Haque MS ，Islam MS ，You MJ 《-》