Hemolymph defensin from the hard tick Haemaphysalis longicornis attacks Gram-positive bacteria.

Ticks are key vectors of some important diseases of humans and animals. Although they are carriers of disease agents, the viability and development of ticks are not harmed by the infectious agents due to their innate immunity. Antimicrobial peptides directly protect hosts against pathogenic agents such as viruses, bacteria, and parasites. Among the identified and characterized antimicrobial peptides, defensins have been considerably well studied. Defensins are commonly found among fungi, plants, invertebrates, and vertebrates. The sequence of the tick hemolymph defensin (HEdefensin) gene from the hard tick Haemaphysalis longicornis was analyzed after identification and cloning from a cDNA library. HEdefensin has a predicted molecular mass of 8.15 kDa including signal peptides and a theoretical isoelectric point of 9.48. Six cysteine residues were also identified in the amino acids. The synthetic HEdefensin peptide only showed antibacterial activity against Gram-positive bacteria such as Micrococcus luteus. A fluorescence propidium iodide exclusion assay also showed that HEdefensin increased the membrane permeability of M. luteus. Additionally, an indirect fluorescent antibody test showed that HEdefensin binds to M. luteus. These results suggested that HEdefensin strongly affects the innate immunity of ticks against Gram-positive bacteria.

Characterization and antiviral activity of a newly identified defensin-like peptide, HEdefensin, in the hard tick Haemaphysalis longicornis.

Tick defensins are antimicrobial peptides that play a major role in the innate immunity of ticks by providing a direct antimicrobial defense. In this study, we identified and characterized a defensin-like encoding gene, HEdefensin, from the expressed sequence tags (EST) database of hemolymph from the hard tick Haemaphysalis longicornis. Expression of the gene in whole adult ticks and in different organs was upregulated during blood feeding, though not after Langat virus (LGTV) challenge. A synthetic HEdefensin peptide demonstrated significant virucidal activity against LGTV but not against an adenovirus in co-incubation virucidal assays. Moreover, the RNAi-mediated gene silencing of HEdefensin did not significantly affect the virus titer as compared to the control group. The data reported here have established the in vitro virucidal activity of the peptide against LGTV. However, its role in the innate antiviral immunity of H. longicornis remains to be explored, and further studies are needed to fully evaluate the potential biological activities of the peptide against bacteria, fungi or parasites.

Improvement of the cryopreservation method for the Babesia gibsoni parasite by using commercial freezing media.

In vitro cultivation and cryopreservation under liquid nitrogen have already been reported and established for Babesia bovis and Babesia bigemina parasites. Although the in vitro cultivation methods for Babesia gibsoni have been reported and established, the cryopreservation methods for this parasite have not been established completely. In this paper, we compared several freezing media for the cryopreservation of B. gibsoni parasite. The CELLBANKER® series (1 plus and 2), STEM-CELLBANKER®, and CultureSure® were used for commercial freezing media; 10% dimethyl sulfoxide in 90% fetal bovine serum, 20% polyvinylpyrrolidone in phosphate-buffered saline (established for bovine Babesia parasites), and 28% glycerol supplemented with 3% sorbitol and 0.65% NaCl dissolved in water (established for Plasmodium parasites) were used for conventional media. Our results demonstrated that the CELLBANKER® series (1 plus and 2), STEM-CELLBANKER®, and CultureSure® are effective freezing media for B. gibsoni parasite compared to the cryopreservation methods of bovine Babesia and Plasmodium parasites. Our improved method of cryopreservation would contribute to the stability of the in vitro cultivation of B. gibsoni parasite.