Enzymatic characterization of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis

In the present study, we performed enzymatic characterization of Haemaphysalis longicornis serine proteinase (HlSP) with a view to shed light on the mechanisms of blood digestion in the hard ticks. Escherichia coli-expressed recombinant HlSP (rHlSP) was shown to potently hydrolyze the synthetic substrates Bz-(DL)-Arg-pNA, Z-Ala-Ala-Leu-pNA and Suc-Ala-Ala-Ala-pNA and yielded an activity of 31.5, 88.2 and 18.3 mumol/min/mg protein, respectively at an optimum temperature of 25 degrees C. However, the enzyme showed little activity to hydrolyze the substrates Suc-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-MCA and Pyr-Phe-Leu-pNA. The optimum pH for the enzyme was shown to be 4.0 to 5.0. Several inhibitors such as antipain, leupeptin and phenylmethylsulfonyl fluoride (PMSF), specific for serine proteinase were shown to inhibit enzyme activity by 20-82%, while E-64 (specific for cysteine proteinases) and pepstatinA (specific for aspartic proteinases) had shown only little inhibitory effects on it. This is the first report on enzymatic characterization of a functional serine proteinase from the hard ticks.     

Molecular cloning of two caspase-like genes from the hard tick Haemaphysalis longicornis

We identified two caspase-like genes from the midgut cDNA library of the hard tick, Haemaphysalis longicornis. Sequence analysis showed that these cDNAs encoded homologues of caspase-2 and caspase-8 that were categorized as apoptosis initiators. The H. longicornis caspase-2 (Hlcaspase-2) cDNA encodes 340 amino acid residues with a predicted molecular weight (Mw) of 38.5 kDa. Another cDNA identified as the H. longicornis caspase-8 (Hlcaspase-8) encodes 306 amino acid residues with an estimated Mw of 35.3 kDa. A catalytic active site was highly conserved in Hlcaspase-8 but not in Hlcaspase-2. RT-PCR analysis showed that both Hlcaspase-2 and Hlcaspase-8 were expressed in tick midgut and salivary glands. This is the first report of the molecular cloning of apoptosis-related genes in the tick.     

Gene silencing of a cubilin-related serine proteinase from the hard tick Haemaphysalis longicornis by RNA interference

RNA interference (RNAi) has been recently exploited to determine gene function by degrading specific mRNAs in several eukaryotic organisms. We constructed a double stranded RNA (dsRNA) from a previously cloned Haemaphysalis longicornis serine proteinase (HlSP) gene to test the importance of the function of the HlSP gene product during blood-feeding. Growth of unfed ticks treated with HlSP dsRNA was significantly inhibited compared to that of PBS-treated ticks. This inhibition was supported by the level of HlSP mRNA. HlSP may play a crucial role for blood-feeding in these ticks. This is the first report on gene silencing of a functional serine proteinase in hard ticks.     

Production and characterization of monoclonal antibodies against midgut of ixodid tick,Haemaphysalis longicornis

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.     

Molecular cloning, expression and characterization of a functional GSTmu class from the cattle tick Boophilus annulatus

A full-length cDNA of a glutathione S-transferase (GST) was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. The 672 bp cloned fragment was sequenced and showed an open reading frame encoding a protein of 223 amino acids. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the sequence is closely related to the mammalian mu-class GST. The cloned gene was expressed in E. coli under T7 promotor of pET-30b vector, and purified under native conditions. The purified enzyme appeared as a single band on 12% SDS-PAGE and has a molecular weight of 30.8 kDa including the histidine tag of the vector. The purified enzyme was assayed upon the chromogenic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the recombinant enzyme showed high level of activity even in the presence of the beta-galactosidase region on its 5' end and showed maximum activity at pH 7.5. The Km values for CDNB and GSH were 0.57 and 0.79 mM, respectively. The over expressed rBaGST showed high activity toward CDNB (121 units/mg protein) and less toward DCNB (29.3 units/mg protein). rBaGST exhibited peroxidatic activity on cumene hydroperoxide sharing this property with GSTs belonging to the GST alpha class. I50 values for cibacron blue and bromosulfophthalein were 0.22 and 8.45 microM, respectively, sharing this property with the mammalian GSTmu class. Immunoblotting revealed the presence of the GST molecule in B. annulatus protein extracts; whole tick, larvae, gut, salivary gland and ovary. Homologues to the GSTmu were also detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. while in Ornithodoros moubata, GSTmu homologue could not be detected.     

Development of Babesia ovata in the midgut of the tick, Haemaphysalis longicornis

Studies were made on the development of Babesia ovata in the midgut of the nymphal tick vector, Haemaphysalis longicornis. In 12 hr post-repletion, merozoites were observed outside of erythrocytes infected with B. ovata in the contents of the midgut of the tick. After that, these merozoites were transformed into ring-forms which were comparatively large ring 2-3 microns in diameter. Within 48-72 hr post-repletion, ring-form protozoa developed into spherical form 4-5 microns in diameter. Within 3-4 days post-repletion, fission-forms were transformed into fission-bodies 2-3 microns in diameter. Within 4-6 days post-repletion, fission-bodies developed into bizarre-forms 6-7 microns in diameter. At this time, elongated form protozoa which were considered as microgametes, 6-8 microns in length, are also seen. Within 6-8 days post-repletion, round-formed protozoa which were considered as zygotes in 9-10 microns in diameter were observed in the gut. About 10 days after repletion, those round-formed protozoa were transformed into vermicule-formed and round-formed protozoa, 13-15 microns in length, appeared again in the gut epithelial cells.     

Molecular cloning and expression of a larval immunogenic protein from the cattle tick Boophilus annulatus

A full-length cDNA of an immunogenic protein was cloned from a cDNA library of the local Egyptian cattle tick Boophilus annulatus. Antibodies raised against B. annulatus larval proteins were used to screen a cDNA expression library. A 936bp cloned fragment was sequenced and showed an open reading frame of 516bp encoding a protein of 171 amino acids. Comparison of the deduced amino acid sequence with protein data bank revealed that the sequence is related to a sequence isolated from the hard tick Haemaphysalis qinghaiensis (Hq05). Southern blot analysis of B. annulatus genomic DNA showed that the cloned cDNA hybridized to double bands per restriction digest, suggesting that the cloned cDNA is a double copy gene. Amino acid analysis of the cloned gene revealed the presence of two casein kinase II phosphorylation sites in the N-terminal domain suggesting that this molecule may be involved in the signal transduction or gene expression pathways. RT-PCR and northern blotting revealed the presence of two isoforms of the Ba05 gene in salivary glands and in the 3-day-old eggs. The cloned gene without the signal peptide, was expressed in Escherichia coli under T7 promotor of pET-30b vector, and purified under denaturation conditions. The purified protein appeared as a single band on 12% SDS-PAGE with a molecular weight around 22.8kDa including the histidine tag of the vector. Antibodies raised against the purified molecule were used to detect the B. annulatus homologue to the Hq05 gene in whole tick, larvae and gut protein extracts. Immunoblotting revealed the presence of this molecule Ba05 only in whole tick and larval protein extracts and not in the gut protein extract. Using the same antibodies, homologues to the Ba05 gene were detected in other tick species as Hyalomma dromedarii and Rhipicephalus sp. but not in Ornithodoros moubata.     

Detection of a spotted fever group Rickettsia in the tick Haemaphysalis juxtakochi in Rondonia, Brazil

The tick genus Haemaphysalis is represented by four species in the New World, of which only the species Haemaphysalis leporispalustris has been associated with Rickettsiae. The present study reports for the first time the presence of a Rickettsia strain in the tick Haemaphysalis juxtakochi. A free-living male of H. juxtakochi, collected in the state of Rondonia, Western Amazon, Brazil, was subjected to DNA extraction and tested by PCR targeting the four rickettsial genes: gltA, 17-kDa, ompA and ompB. The nucleotide sequences obtained from the PCR products were, by BLAST analyses, closest to Rickettsia rhipicephali sharing 99.7% (1147/1150), 98.8% (429/434), 99.0% (486/491) and 99.0% (809/817) identities with the partial sequences of the gltA, 17-kDa, ompA and ompB genes, respectively. Phylogenetic analyses inferred from the four rickettsial genes showed a high-degree of similarity of this H. juxtakochi-Rickettsia with R. rhipicephali. These two agents grouped together in all trees, always with high bootstrap support (75-96%). This study gives molecular evidence for the presence of a Rickettsia species, designated as strain R300, in the tick H. juxtakochi from the Western Amazon area of Brazil. Genetic analyses showed R300 to be closely related to R. rhipicephali.     

镰形扇头蜱肌钙蛋白I基因的克隆及其分布

本试验从镰形扇头蜱半饱血雌蜱唾液腺cDNA文库的EST中筛选了1个含polyA尾的基因序列,经5LRACE方法得到该基因全长序列.经同源性比较,该基因预测的氨基酸序列与长角血蜱肌钙蛋白I(GI14041807)的同源性为84.47%,肌动蛋白结合位点位于147-167氨基酸处,且与长角血蜱肌钙蛋白I肌动蛋白结合位点完全相同,表明该基因是镰形扇头蜱肌钙蛋白I基因.以镰形扇头蜱基因组DNA为模板扩增到编码肌钙蛋白I的基因组序列.序列分析表明该序列不含内含子.RT-PCR分析表明该基因在镰形扇头蜱的卵及其幼蜱、若蜱、成蜱的壳、唾液腺和肠均有表达.     

Molecular evidence of Babesia caballi (Nuttall and Strickland, 1910) parasite transmission from experimentally-infected SCID mice to the ixodid tick, Haemaphysalis longicornis (Neuman, 1901).

Molecular evidence that suggests the possible role of the ixodid tick, Haemaphysalis longicornis and its eggs in the transmission of equine Babesia caballi parasites is presented herein. Using polymerase chain reaction (PCR) to assay for DNA in parasites, presumably acquired by ticks that were allowed to feed on splenectomized-SCID mice, experimentally exposed to in vitro-cultivated B. caballi, we have obtained positive bands that corresponded to the expected B. caballi-specific 430bp gene fragment in 50% of female ticks used, and in 75 and 25% of eggs and larval progeny, respectively. Also, parasite DNA was detected in ticks, eggs and larvae as late as the 16th to the 20th day post-host infestation. Present findings support to the potential role of H. longicornis in the transmission of B. caballi parasites. Its capability, however, to successfully transmit the infection to horses under natural conditions in the field needs to be further ascertained. To our knowledge, this is the first documented study incriminating H. longicornis as a most and likely biological vector of equine babesias.     

First glimpse into the origin and spread of the Asian longhorned tick,Haemaphysalis longicornis,in the United States

Established populations of Asian longhorned ticks (ALT), Haemaphysalis longicornis, were first identified in the United States (US) in 2017 by sequencing the mitochondrial cytochrome c oxidase subunit I (cox1) ‘barcoding’ locus followed by morphological confirmation. Subsequent investigations detected ALT infestations in 12, mostly eastern, US states. To gain information on the origin and spread of US ALT, we (1) sequenced cox1 from ALT populations across 9 US states and (2) obtained cox1 sequences from potential source populations [China, Japan and Republic of Korea (ROK) as well as Australia, New Zealand and the Kingdom of Tonga (KOT)] both by sequencing and by downloading publicly available sequences in NCBI GenBank. Additionally, we conducted epidemiological investigations of properties near its initial detection locale in Hunterdon County, NJ, as well as a broader risk analysis for importation of ectoparasites into the area. In eastern Asian populations (China/Japan/ROK), we detected 35 cox1 haplotypes that neatly clustered into two clades with known bisexual versus parthenogenetic phenotypes. In Australia/New Zealand/KOT, we detected 10 cox1 haplotypes all falling within the parthenogenetic cluster. In the United States, we detected three differentially distributed cox1 haplotypes from the parthenogenetic cluster, supporting phenotypic evidence that US ALT are parthenogenetic. While none of the source populations examined had all three US cox1 haplotypes, a phylogeographic network analysis supports a northeast Asian source for the US populations. Within the United States, epidemiological investigations indicate ALT can be moved long distances by human transport of animals, such as horses and dogs, with smaller scale movements on wildlife. These results have relevant implications for efforts aimed at minimizing the spread of ALT in the United States and preventing additional exotic tick introductions.     

Purification and characterization of a 45-kDa concealed antigen from the midgut membranes of Ornithodoros erraticus that induces lethal anti-tick immune responses in pigs

Ornithodoros erraticus is an argasid tick that can transmit severe diseases such as human relapsing fever and African swine fever. In the search for a vaccine against this parasite, a crude extract of tick midgut membranes (GME) was obtained that in pigs and mice induced a protective response able to kill up to 80% of the nymphs in the first 72 h post-feeding and to reduce the fecundity of females by more than 50%. To identify the protective antigens, the GME was subjected to successive biochemical fractionations and the resulting simpler protein fractions were inoculated in pigs. A 45-kDa antigen, the so-called Oe45, was detected, purified and demonstrated to be responsible for the protection induced by the GME. Oe45 seems to be a membrane protein that is presumably expressed on the luminal membrane of midgut epithelial cells. Oe45 consists of at least two differently charged bands (cationic and neutral), which show antigenic cross-reactivity. The possibility that these bands might be different isoforms of the same protein is discussed. Although Oe45 is constitutively expressed at low levels throughout the trophogonic cycle, its expression is up-regulated by the ingestion of blood, as suggested by the higher levels observed between 6 and 72 h post-feeding.