Iron regulatory protein from the hard tick Haemaphysalis longicornis: characterization, function and assessment as a protective antigen.

BACKGROUND: Ticks are blood-feeding ectoparasites with different host specificities and are capable of pathogen transmission. Iron regulatory proteins (IRPs) play crucial roles in iron homeostasis in vertebrates. However, their functions in ticks remain poorly understood. The aim of the present study was to investigate the characteristics, functions, molecular mechanisms, and the vaccine efficacy of IRP in the hard tick Haemaphysalis longicornis. RESULTS: The full-length complementary DNA of IRP from Haemaphysalis longicornis (HlIRP) was 2973 bp, including a 2772 bp open reading frame. It is expressed throughout three developmental stages (larvae, nymphs, and adult females) and in various tissues (salivary glands, ovaries, midgut, and Malpighian tubules). Recombinant Haemaphysalis longicornis IRP (rHlIRP) was obtained via a prokaryotic expression system and exhibited aconitase, iron chelation, radical-scavenging, and hemolytic activities in vitro. RNA interference-mediated IRP knockdown reduced tick engorgement weight, ovary weight, egg mass weight, egg hatching rate, and ovary vitellin content, as well as prolonging the egg incubation period. Proteomics revealed that IRP may affect tick reproduction and development through proteasome pathway-associated, ribosomal, reproduction-related, and iron metabolism-related proteins. A trial on rabbits against adult Haemaphysalis longicornis infestation demonstrated that rHlIRP vaccine could significantly decrease engorged weight (by 10%), egg mass weight (by 16%) and eggs hatching rate (by 22%) of ticks. The overall immunization efficacy using rHlIRP against adult females was 41%. CONCLUSION: IRP could limit reproduction and development in Haemaphysalis longicornis, and HlIRP was confirmed as a candidate vaccine antigen to impair tick iron metabolism and protect the host against tick infestation. © 2024 Society of Chemical Industry.

Molecular characterization of Hyalomma dromedarii and evaluation of acaricidal potential of herbal methanolic extracts against H. dromedarii larvae in comparison to synthetic acaricides.

The present study had two aims: molecular characterization of Hyalomma dromedarii infesting one-humped camels of Haryana (North India), and assessment of the acaricidal potential of herbal methanolic extracts against H. dromedarii larvae in comparison to synthetic acaricides. Phylogenetics and population neutrality indices were assessed by targeting partial amplification of mitochondrial 16S rDNA sequences. Larval packet test (LPT) was performed to evaluate the acaricidal efficacy of herbal extracts (Ferula asafoetida and Trachyspermum ammi) and synthetic acaricides (deltamethrin and fipronil). Phylogenetic studies established the collected ticks to be H. dromedarii, exhibiting a homology of 99.8-100%. However, the present study isolates formed a different sub-clade compared to H. dromedarii sequences from Egypt, Senegal, Tunisia and Saudi Arabia. Nucleotide and haplotype diversity values were indicative of demographic expansion and low gene flow. Negative values of Tajima's D (-0.612) and Fu and Li's Fst (-0.479) highlighted deviations from neutrality and emphasized recent population expansion. The median lethal concentration (LC50) values recorded for T. ammi, F. asafoetida and their combination were 3.68, 2.87 and 2.59 mg/mL, respectively, whereas the 90% lethal concentration (LC90) values were 4.09, 3.58 and 3.35 mg/mL, respectively. It was also observed that the H. dromedarii population under study was completely susceptible to both the formulated synthetic acaricides. In conclusion, combination of methanolic extracts of F. asafoetida and T. ammi could provide a potential substitute to toxic synthetic chemical acaricides and might prove a valuable component of integrated tick management strategies.

Evaluation on two types of paramyosin vaccines for the control of Haemaphysalis longicornis infestations in rabbits.

BACKGROUND: Haemaphysalis longicornis is an obligate hematophagous ectoparasite that transmits a variety of pathogens causing life-threatening diseases in humans and animals. Paramyosin (Pmy) is not only an invertebrate-specific myofibrillar protein but also an important immunomodulatory protein. Therefore, it is one of the ideal candidate antigens for vaccines. METHODS: We conducted two vaccine trials to evaluate the protective efficacy of Pmy recombinant protein (rPmy) and peptide vaccine (KLH-LEE). Each rabbit was immunized with three doses of rPmy or KLH-LEE adjuvanted with Freund's complete/incomplete at 500 µg/dose at 2-week intervals before challenge with 40 female H. longicornis/rabbit. PBS plus adjuvant, Trx or KLH was used as control group. The antibodies of rabbits were detected by ELISA. Then, female ticks were fed on the rabbits until detachment. RESULTS: ELISA results showed that both vaccines induced rabbits to produce antibodies. Compared with the Trx group, the engorgement weight, oviposition and hatchability of the rPmy group decreased by 8.87%, 26.83% and 38.86%, respectively. On the other hand, engorgement weight, oviposition and hatchability of female ticks in the KLH-LEE group correspondingly resulted in 27.03%, 53.15% and 38.40% reduction compared with that of the KLH group. Considering the cumulative effect of vaccination on the evaluated parameters, results showed 60.37% efficacy of the rPmy vaccine formulation and 70.86% efficacy in the KLH-LEE group. CONCLUSIONS: Pmy and particularly epitope LEE have potential for further development of an effective candidate vaccine to protect the host against tick infection. GRAPHIC ABSTARCT.

Molecular characterization of an alanine-, proline-, glycine-, threonine-, and serine-rich protein of the hard tick Haemaphysalis qinghaiensis and its effect as a vaccine against tick infestation in sheep.

An immunological tick control approach has been proved to be the most promising alternative strategy compared to the current usage of acaricides that suffers from a number of serious limitations. The success of this method is mainly dependent on the identification of tick antigen candidates. Here, the complete sequence of a positive clone Hq15 that we screened from a cDNA library of Haemaphysalis qinghaiensis was cloned by using rapid amplification of cDNA ends. Hq15 contains an open reading frame (ORF) of 1851 bp that codes for 616 amino acid residues with a coding capacity of 61 kDa. The deduced Hq15 amino acid sequence was characterized by a high content of alanine (13.80%), proline (12.82%), glycine (12.18%), threonine (10.71%), and serine (10.06%). Sequence similarity and phylogenetic analyses indicated that Hq15 might encode a novel protein of the tick. Expression analysis by reverse transcription polymerase chain reaction indicated that the gene was expressed in every developmental stage of the tick in its salivary glands, but not in the midgut. The cDNA was expressed as glutathione S-transferase-fused protein in a prokaryotic system. Vaccination of sheep with rHq15 conferred a significant protective immunity in sheep, resulting in a reduction of the amount of eggs laid by each tick compared to the controls. These results show that rHq15 might be one of the candidate vaccine molecules for the control of ticks.

Multiple ferritins are vital to successful blood feeding and reproduction of the hard tick Haemaphysalis longicornis.

Ticks are obligate hematophagous parasites and important vectors of diseases. The large amount of blood they consume contains great quantities of iron, an essential but also toxic element. The function of ferritin, an iron storage protein, and iron metabolism in ticks need to be further elucidated. Here, we investigated the function a newly identified secreted ferritin from the hard tick Haemaphysalis longicornis (HlFER2), together with the previously identified intracellular ferritin (HlFER1). Recombinant ferritins, expressed in Escherichia coli, were used for anti-serum preparation and were also assayed for iron-binding activity. RT-PCR and western blot analyses of different organs and developmental stages of the tick during blood feeding were performed. The localization of ferritins in different organs was demonstrated through an indirect immunofluorescent antibody test. RNA interference (RNAi) was performed to evaluate the importance of ferritin in blood feeding and reproduction of ticks. The midgut was also examined after RNAi using light and transmission electron microscopy. RT-PCR showed differences in gene expression in some organs and developmental stages. Interestingly, only HlFER2 was detected in the ovary during oviposition and in the egg despite the low mRNA transcript. RNAi induced a reduction in post-blood meal body weight, high mortality and decreased fecundity. The expression of vitellogenin genes was affected by silencing of ferritin. Abnormalities in digestive cells, including disrupted microvilli, and alteration of digestive activity were also observed. Taken altogether, our results show that the iron storage and protective functions of ferritin are crucial to successful blood feeding and reproduction of H. longicornis.

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.

Cloning and characterization of a cDNA clone encoding calreticulin from Haemaphysalis qinghaiensis (Acari: Ixodidae).

The application of anti-tick vaccine has been shown to be the most promising alternative strategy compared to the current use of acaricides that suffer from a number of serious limitations. The success of this method is dependent upon identification and cloning of potential tick vaccine antigens. Previously, we have cloned 21 positive clones (named from Hq02 to Hq22) by immunoscreening complimentary DNA (cDNA) libraries of Haemaphysalis qinghaiensis; however, some of those clones did not contain open reading frames (ORF). In this study, we amplified the entire sequence of Hq07 by using rapid amplification of the cDNA ends. Hq07 contains an ORF of 1,233 bp that encodes for 410 amino acid residues with a coding capacity of 47 kDa. Search of the cloned sequences against GenBank revealed that Hq07 is a calreticulin (CRT)-similar clone and designated HqCRT. Expression analysis by reverse transcription-polymerase chain reaction showed that this gene is ubiquitously expressed at different developmental stages and in different tissues of H. qinghaiensis. The gene was expressed as glutathione S-transferase-fused proteins in a prokaryotic system. Western blot analysis revealed that native HqCRT was secreted into their hosts by ticks during blood sucking. Vaccination of sheep with rHqCRT conferred protective immunity against ticks, resulting in 54.3% mortality in adult ticks, compared to the 38.7% death rate in the control group. These results demonstrated that rHqCRT might be a useful vaccine candidate antigen for biological control of H. qinghaiensis.

Baculovirus expression of BmAChE3, a cDNA encoding an acetylcholinesterase of Boophilus microplus (Acari: Ixodidae).

The complete cDNA sequence encoding a Boophilus microplus (Canestrini) (Acari: Ixodidae) acetylcholinesterase (AChE3) was expressed in the baculovirus system. The recombinant AChE3 protein (rBmAChE3) was secreted as a soluble form into the cell culture medium and was identified as a functional AChE by substrate specificity and by inhibition with the AChE-specific inhibitors eserine sulfate and BW284c51. Inhibition kinetics of rBmAChE3, in the presence of the organophosphate paraoxon, revealed sensitivity comparable with that of adult, organophosphate-susceptible neural AChE. To our knowledge, this is the first report of the cloning and successful expression of a functional ixodid AChE.

The molecular and biological analysis of ixodid ticks histamine release factors.

We previously described a Dermacentor varibialis (DV) cDNA that encodes a ubiquitously expressed and tick saliva-secreted functional histamine release factor (HRF) homolog. In this study gene specific primers based on DVHRF open reading frame nucleotide sequence were utilized to amplify three orthologs, from the wood tick, D. andersoni (DA), the black legged tick, the southern cattle tick, Boophilus microplus (BM) and the lone star tick, Amblyomma americanum (AA). At nucleotide level, sequence comparisons revealed 98 89 and 84% similarity to DVHRF for DAHRF, AAHRF and BMHRF, respectively, while predicted polypeptide comparisons revealed 98, 96 and 91% similarity for DAHRF, AAHRF and BMHRF respectively. Phylogenetically, the tick HRF clade, while distinct (100% bootstrap value), is closely related to other arthropods, but distantly related to vertebrate and protozoan clades. Consistent with sequence similarity analysis, a DVHRF-specific northern blotting probe hybridized a approximately 900 base pair (bp) mRNA band on all RNA blots. Likewise a mouse polyclonal antibody to E. coli-expressed recombinant (r) DVHRF, cross-reacted baculovirus-expressed non-fusion rAAHRF, rDAHRF, and rBMHRF. As revealed by northern blotting analysis of larvae and nymph RNA, DVHRF mRNA is expressed in both immature and mature ticks indicating that its transcription is not developmentally regulated. Unlike rHRF/TCTP proteins of other organisms, the calcium-binding function may not be conserved for tick HRF homologs as revealed by the 45CaCl2+ overlay assay. Apparent global expression of DVHRF and its orthologs make this protein family an ideal target antigen for development of novel tick control strategies targeting multiple tick species.

Hard tick calreticulin (CRT) gene coding regions have only one intron with conserved positions and variable sizes.

Calreticulin (CRT) is a unique eukaryotic gene. The CRT gene product, calreticulin, was first identified as a calcium binding protein in 1974, but further investigations have indicated that CRT protein performs many functions in cells, including involvement in evading the host's immune system by parasites. Many studies of CRT have been published since the molecule was first discovered; however, the CRT gene exon-intron structure is only known for a limited number of ectoparasite species. In this study, we compared tick CRT genomic sequences to the corresponding cDNA from 28 species and found that 2 exons and 1 intron are present in the tick CRT gene. The intron position is conserved in 28 hard ticks, but intron size and nucleotide sequences vary. Three tick introns possess duplicated fragments and are twice as long as other introns. All tick CRT introns obey the GT-AG rule in the splice-site junctions and are phase 1 introns. By comparing tick CRT introns to those of fruit fly, mouse, and human, we conclude that tick CRT introns belong to the intron-late type. The number and size of CRT introns have increased through the evolution of eukaryotes.

Identification of a third Boophilus microplus (Acari: Ixodidae) cDNA presumptively encoding an acetylcholinesterase.

Oligodeoxynucleotide primers, based on amino acid sequences conserved in known acetylcholinesterases (AChEs), were used in reverse-transcription polymerase chain reaction (RT-PCR) with mRNA from Boophilus microplus (Canestrini) as the template. Primer walking and rapid amplification of cDNA ends (RACE) techniques were used to complete the cDNA sequence identified by RT-PCR. The complete B. microplus cDNA sequence contained an open reading frame encoding a 620 amino acid protein with a 20 amino acid signal peptide at the N-terminus targeting the protein for the secretion pathway. BLAST searches of GenBank using the presumptively encoded protein revealed highest sequence similarity to AChEs. The presumptively encoded protein was of similar size and structural properties to other identified AChEs, including the presence of the catalytic triad (Ser, Glu, His) and appropriate placement of internal cysteines to yield three internal disulfide bonds corresponding to those of known AChEs. Putative conserved domains identified the sequence as a member of the carboxylesterase family, pfam00135.8, of which AChE is a member. This cDNA therefore presumptively encodes a third transcribed AChE (AChE3) cDNA of B. microplus. Comparison of the three AChE eDNA sequences expressed in B. microplus demonstrated no discernible nucleotide sequence homology and relatively low amino acid sequence homology, strongly suggesting that they are not alleles of one another. The potential presence of multiple expressed AChEs in B. microplus suggests alternative mechanisms for development of resistance to pesticides that target AChE. The homology-based identification of a third expressed AChE in B. microplus is a surprising result and strongly implies the need for confirmation of gene identity for presumptive AChEs.

Cloning and sequencing of putative calreticulin complementary DNAs from four hard tick species.

Calreticulin (CRT) is a calcium-binding protein and has many functions in eukaryotic cells. CRT is possibly involved in parasite host immune system evasion. To better understand the molecular basis of CRT in ticks, we cloned and sequenced 4 full-length complementary DNAs (cDNAs) from the hard tick species, Dermacentor variabilis, Haemaphysalis longicornis, Ixodes scapularis, and Rhipicephalus sanguineus, using the technique of rapid amplification of cDNA ends. The deduced amino acid sequences share high identities (between 77 and 98%) with 3 known tick CRT sequences. The major characteristics of known CRTs are observed in all 4 of our deduced tick CRTs. These include 3 major domains, a signal peptide sequence at the beginning of the coding region, 2 triplets of conserved regions, cysteine sites providing disulfide bridges for N-terminal folding, and a nuclear localization signal. Remarkably, the replacement of the endoplasmic reticulum retention signal KDEL by HEEL, which is believed to be associated with secretion of CRT into the host during feeding and was previously recorded only in 2 ticks and a hookworm, is also present in all 4 of our tick putative CRTs. In addition, the CRT gene is potentially useful for tick phylogenetic reconstruction.

Cloning and sequencing of putative acetylcholinesterase cDNAs from the American dog tick, Dermacentor variabilis, and the brown dog tick, Rhipicephalus sanguineus (Acari: Ixodidae).

Two putative cDNAs of acetylcholinesterase (AChE), one from Dermacentor variabilis, and the other from Rhipicephalus sanguineus, were amplified and sequenced. The deduced amino acid sequences have high amino acid identities (between 70 and 94%) to known tick AChE sequences deposited in GenBank. Furthermore, these two AChEs also possess common features in their primary AChE structure such as catalytic active sites. A 2,220-bp contiguous sequence, containing a 1,791-bp open reading frame encoding an AChE precursor with 596 amino acid residues, was obtained from D. variabilis. The deduced proteins of R. sanguineus are different in size by 6 amino acids because of alternative splicing at the 5' end. A gene tree deduced from phylogenetic analysis indicates that there are at least three lineages of AChE in arthropods.

Cloning and partial characterization of a Boophilus microplus (Acari: Ixodidae) calreticulin.

We report the cloning, sequence characterization and expression analysis of a calreticulin (CRT) coding cDNA of Boophilus microplus. CRT is a calcium-binding protein involved in multiple cell functions and possibly implicated in parasites host immune system evasion. The CRT cDNA sequence and its molecular characterization are described. Sequence similarity and phylogenetic analyses indicate a close relationship to other arthropod CRT sequences. The CRT cDNA was also expressed in a procariotic system and the recombinant protein (rBmCRT) was used to raise antibodies in a rabbit. Expression analyses of the corresponding gene in different developmental stages and tissues were performed by RT-PCR and Western-blot, which indicated a ubiquitous expression of the B. microplus calreticulin gene and demonstrated its presence in saliva. Sera of tick-infested bovines suggested that this protein may not be able to induce an IgG-based humoral response in its natural host.