Saiki Imamura

A serine proteinase inhibitor (serpin) from ixodid tick Haemaphysalis longicornis; cloning and preliminary assessment of its suitability as a candidate for a tick vaccine

Rapid amplification of the cDNA ends (RACE) and primers designed based on a conserved serpin amino acid motif (NAVYFKG) were used to clone a 1350bp cDNA which encodes a 378 polypeptide with high sequence similarity to several known serpins. We have named this gene as Haemaphysalis longicornis serpin-1 (HLS1). Northern blotting and reverse transcription (RT)-PCR analysis of total RNA from unfed or partially fed whole ticks as well as dissected tick organs revealed that transcription of HLS1 mRNA was induced by blood meal feeding during the slow feeding phase (24-48 h post-attachment) only in the tick midguts. Vaccination of rabbits with recombinant HLS1 (rHLS1) expressed in Escherichia coli resulted in 43.9 and 11.2% mortality of nymph and adult ticks which were fed on immunized rabbits. Polyclonal rabbit antibodies to tick saliva did not react with rHLS1, suggesting that native HLS1 was not secreted into the host during tick feeding. rHLS1 could be a potential candidate for a cocktail anti-tick vaccine.

Cross immunity with Haemaphysalis longicornis glutathione S-transferase reduces an experimental Rhipicephalus (Boophilus) microplus infestation

Recombinant Glutathione S-transferase of Haemaphysalis longicornis (rGST-Hl) was expressed in Escherichia coli, purified by affinity chromatography and used in the immunization of cattle. Western blot analysis showed positive antibody response in cattle immunized with rGST-Hl. The tests also showed that immunized bovine sera recognize native Rhipicephalus microplus proteins in different tissue extracts. Furthermore, the vaccine potential of rGST-Hl was investigated against infestation of Hereford cattle by R. microplus. Vaccination of cattle with rGST-Hl conferred partial cross-protection immunity against R. microplus. Considering the effect on number of engorged ticks, egg laying capacity and egg fertility, the overall efficacy of vaccination was of 57%, as compared with control group.

Cloning, expression and partial characterization of a Haemaphysalis longicornis and a Rhipicephalus appendiculatus glutathione S-transferase.

The ticks Haemaphysalis longicornis and Rhipicephalus appendiculatus are important parasites worldwide. The current method for control of cattle ticks involves the use of chemicals. Nevertheless, parasite resistance is an ever increasing global problem. Glutathione S‐transferases (GSTs) play a central role in detoxication of xenobiotic and endogenous compounds. Several authors have noted that an increase in GST activity is associated with resistance to insecticides and acaricides. In the present study, we report the cloning and expression of GST cDNAs from H. longicornis and R. appendiculatus. In addition, we determine the effect of three acaricides (ethion, deltamethrin and diazinon) on the enzymatic activity of rGSTs.

Development of a SYBR Green Real-Time Polymerase Chain Reaction Assay for Quantitative Detection of Babesia Gibsoni (Asian Genotype) DNA

A real-time fluorogenic polymerase chain reaction (PCR) assay based on SYBR green that allows for sensitive, reproducible, and accurate quantification of Babesia gibsoni (Asian genotype). DNA from peripheral blood of infected dogs was developed. Standard curves were created by plotting the input amount of a standard template, constructed with plasmid DNA containing 182 base pairs (bp) of the p18 gene, against threshold cycle numbers. The curves showed a wide dynamic range (1,000,000-fold input) and high correlation values (>0.99). The PCR amplification efficacy of the standard template was similar to that of intact genomic DNA obtained from peripheral blood with B. gibsoni infection. The detection limit of the assay was 9 parasites/μl of blood with B gibsoni infection. The intra-assay and interassay coefficients of variation of the threshold cycles ranged from 0.70% to 1.89% and from 1.18% to 1.92%, respectively. This assay system was found to be reproducible and accurate for the quantification of parasite DNA in experimentally infected dogs and far more sensitive than traditional microscopic examination.

Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

Ixodes persulcatus is the primary vector for human tick‐borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin‐like antimicrobial peptide was identified. The amino‐acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram‐positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram‐negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

Comparative immunogenicity of Haemaphysalis longicornis and Rhipicephalus (Boophilus) microplus calreticulins.

The ticks Rhipicephalus (Boophilus) microplus and Haemaphysalis longicornis are blood-sucking ectoparasites of bovines, causing serious damages to the livestock production. The main control method for these ticks is based on acaricides. However, the use of vaccines has been studied as a promising control strategy. Calreticulin (CRT) is a multifunctional, predominantly intracellular protein present in almost all cells of animals. The secretion of CRT during feeding might be linked to the modulation of the parasite-host interaction. In the present study, recombinant CRTs of R. microplus (rBmCRT) and H. longicornis (rHlCRT) were expressed in Escherichia coli and purified by ion exchange chromatography and used for the immunization of bovines and mouse. ELISA demonstrated that both rCRTs are recognized by the sera of immunized bovines. In silico, despite the difference in amino acid sequences, antigenic index analysis of HlCRT and BmCRT using the Jameson-Wolf algorithm indicated that both proteins were very similar in antigenicity index, although six different epitopes between the tick CRTs have been inferred. These data were corroborated by competitive ELISA analyses, which suggest the presence of different epitopes within the proteins. Western blot analyses showed that anti-rBmCRT and anti-rHlCRT bovine sera also recognized the native proteins in larvae extracts and, moreover, sera of bovines immunized with saliva and extract of salivary glands recognized both recombinant CRTs. Thus, mouse and bovine immune system recognized rCRTs, resulting in the production of antibodies with similar specificity for both recombinant proteins, although different epitopes could be distinguished between rBmCRT and rHlCRT.

Identification of TROSPA homologue in Ixodes persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan.

The tick receptor for outer surface protein A (TROSPA) is an Ixodes scapularis (I. scapularis) receptor for Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease in North America. The blockade of TROSPA has been shown to reduce B. burgdorferi adherence to the I. scapularis gut in vivo. Thus, TROSPA is one of the potential targets for the development of vector-antigen-based vaccines to prevent the transmission of B. burgdorferi. The aim of this study is to identify the TROSPA gene in I. persulcatus Schulze, the specific vector for human Lyme borreliosis in Japan. The cDNA clone encoding the TROSPA-like sequence with 483 nucleotides was obtained from whole-body homogenates of fed nymphs of I. persulcatus. The putative amino acid sequence of I. persulcatus TROSPA was 88.2% and 87.8% identical to that of I. scapularis and I. ricinus, respectively. This finding will facilitate investigations on the role of I. persulcatus TROSPA and its interaction with Borrelia spp. and will have important implications on endeavors to develop a tick vaccine.

Suppression of cell proliferation and cytokine expression by HL-p36, a tick salivary gland-derived protein of Haemaphysalis longicornis

Previously, a putative immunosuppressant‐coding gene was identified from a complementary DNA library derived from the salivary glands of partially‐fed Haemaphysalis longicornis. Using real‐time polymerase chain reaction, the gene was shown to be predominantly expressed during blood feeding with the site of expression being mainly in the salivary glands; this was confirmed by Western blotting analysis. To investigate the function of this novel protein, in this study, we examined the proliferative responses of bovine mononuclear cells and murine splenic cells as well as the expression of profiles of several cytokines in these cells in the presence of the recombinant protein (H. longicornis‐derived 36 000 molecular weight protein: rHL‐p36). The addition of rHL‐p36 at the beginning of the 72 hr cultivation period clearly inhibited proliferation of several mitogen‐stimulated cells in a dose‐dependent manner, with concomitantly significant down‐regulation of messenger RNA levels for interleukin‐2. The inhibitory response could be abrogated by blockage of HL‐p36 with antibody, suggesting the direct involvement of rHL‐p36 in the cell proliferation. Furthermore, the proliferative response of splenocytes isolated from rHL‐p36‐inoculated mice was significantly lower than for those from control mice, suggesting that rHL‐p36 could also directly suppress immune responses in vivo. Interestingly, microarray analysis of the splenocytes showed that the expression of several immunomodulating genes was down‐regulated by rHL‐p36 inoculation. In conclusion, these results suggest that HL‐p36 is an immunosuppressor that might play an important role in the modulation of host immune responses.

Expression and activity of glycogen synthase kinase during vitellogenesis and embryogenesis of Rhipicephalus (Boophilus) microplus

Glycogen synthase kinase 3 (GSK-3) is classically described as a key enzyme involved in glycogen metabolism in mammals. GSK-3 belongs to a highly conserved family of serine/threonine protein kinases, whose members are involved in hormonal regulation, nuclear signaling, and cell fate determination in higher eukaryotes. We have cloned and characterized the RmGSK-3 gene from Rhipicephalus (Boophilus) microplus tick embryos. DNA and protein sequence analysis depicted high similarity to the corresponding enzyme, from both vertebrate and invertebrate animals. In addition, the mRNA transcription profile identified during embryogenesis was analyzed. We observed that the RmGSK-3 mRNA rapidly decreases from the 1st to 3rd day of development, and increases from the 3rd to 15th day. After the 15th day of development, we observed a near 50% reduction in RmGSK-3 mRNA transcription in comparison to the 1st day. We detected the GSK-3beta isoform in egg homogenates throughout embryogenesis using Western blot analysis. RmGSK-3 mRNA was present in fat body, midgut and ovary from partially and fully engorged adult female ticks. The highest mRNA level was observed in ovaries from both developmental stages and in first-day eggs. Furthermore, RmGSK-3 activity correlated with glycogen content variation. Finally, kinase activity in egg homogenates was inhibited by the specific inhibitor, SB-216763. These data suggest that RmGSK-3beta may be involved in glycogen metabolism regulation during R. microplus embryogenesis.

Effect of vaccination with a recombinant metalloprotease from Haemaphysalis longicornis.

We report the cloning, expression and characterization of an Haemaphysalis longicornis metalloprotease (named HLMP1). The gene encodes a predicted 550 aminoacid protein with similarity to metalloproteases of the reprolysin family. The protein sequence contains a signal sequence, the zinc-binding motif (HEXXHXXGXXH) common to metalloproteases and a cysteine-rich region. Reverse transcription-PCR expression analysis indicates the presence of mRNA in the salivary gland of larva, nymph and adult ticks. Rabbit repeatedly infested with H. longicornis recognized rHLMP1, suggesting that the immune-response against HLMP1 is naturally induced through the feeding of ticks. Vaccination of rabbit with rHLMP1 produced protective immunity against ticks, resulting in 15.6 and 14.6% mortality in nymph and adult ticks, respectively. This work provides information to understand the tick’s defense system, and offers new insights to develop strategies to block this defense system with an anti-tick vaccine based on a metalloprotease.