Tick salivary protein Cystatin: structure, anti-inflammation and molecular mechanism.

Ticks are blood-sucking ectoparasites that secrete immunomodulatory substances in saliva to hosts during engorging. Cystatins, a tick salivary protein and natural inhibitor of Cathepsins, are attracting growing interest globally because of the immunosuppressive activities and the feasibility as an antigen for developing anti-tick vaccines. This review outlines the classification and the structure of tick Cystatins, and focuses on the anti-inflammatory effects and molecular mechanisms. Tick Cystatins can be divided into four families based on structures and cystatin 1 and cystatin 2 are the most abundant. They are injected into hosts during blood feeding and effectively mitigate the host inflammatory response. Mechanically, tick Cystatins exert anti-inflammatory properties through the inhibition of TLR-NF-κb, JAK-STAT and p38 MAPK signaling pathways. Further investigations are crucial to confirm the reduction of inflammation in other cell types like neutrophils and mast cells, and fully elucidate the underlying mechanism (like the structural mechanism) to make Cystatin a potential candidate for the development of novel anti-inflammation agents.

Emerging bacterial infectious diseases/pathogens vectored by human lice.

Human lice have always been a major public health concern due to their vector capacity for louse-borne infectious diseases, like trench fever, louse-borne relapsing fever, and epidemic fever, which are caused by Bartonella quintana, Borrelia recurrentis, and Rickettsia prowazekii, respectively. Those diseases are currently re-emerging in the regions of poor hygiene, social poverty, or wars with life-threatening consequences. These louse-borne diseases have also caused outbreaks among populations in jails and refugee camps. In addition, antibodies and DNAs to those pathogens have been steadily detected in homeless populations. Importantly, more bacterial pathogens have been detected in human lice, and some have been transmitted by human lice in laboratories. Here, we provide a comprehensive review and update on louse-borne infectious diseases/bacterial pathogens.

Egg protein profile and dynamics during embryogenesis in Haemaphysalis flava ticks.

Tick eggs contain all essential proteins for embryogenesis, and egg proteins are a potential reservoir of tick-protective antigens. However, the protein profile and dynamics during embryonic development remain unknown. This study aimed to depict the protein profile and dynamics in tick embryogenesis, further providing protein candidates for targeted interventions. Eggs from Haemaphysalis flava ticks were incubated at 28 °C and 85% relative humidity. On days 0 (newly laid eggs without incubation), 7, 14 and 21, eggs were collected, dewaxed and subject to protein extraction. Extracted proteins were digested by filter-aided sample preparation and analyzed by liquid chromatography-tandem mass spectrometry (LC/MS-MS). MS data were searched against an in-house H. flava protein database for tick-derived protein identification. Abundances of 40 selected high-confidence proteins were further quantified by LC-parallel reaction monitoring (PRM)/MS analysis throughout egg incubation. A total of 93 high-confidence proteins were identified in eggs on 0-day incubation. Identified proteins belonged to seven functional categories: transporters, enzymes, proteinase inhibitors, immunity-related proteins, cytoskeletal proteins, heat shock proteins and uncharacterized proteins. The enzyme category contained the most types of proteins. Neutrophil elastase inhibitors represented the most abundant proteins in terms of intensity-based absolute-protein-quantification. LC-PRM/MS revealed that the abundances of 20 proteins increased including enolase, calreticulin, actin, GAPDH et cetera, and the abundances of 11 proteins decreased including vitellogenins, neutrophil elastase inhibitor, carboxypeptidase Q, et cetera from 0- to 21-day incubation. This study provides the most comprehensive egg protein profile and dynamics during tick embryogenesis. Further investigations are needed to test the tick-control efficacy by targeting the egg proteins.

Saliva proteome of partially- and fully-engorged adult female Haemaphysalis flava ticks.

Tick saliva is a reservoir of bioactive proteins. Saliva protein compositions change dynamically during blood-feeding. Decipherment of protein profiles in different blood-feeding stages may bring deeper insight into tick feeding physiology and provide targets for immunologic control alternatives. However, having the infancy of tick genome sequencing, assembly, annotation, and limited knowledge of tick salivary proteins restrain the data interpretation. Here, we aimed to depict the saliva protein profile in partially- (PE) and fully-engorged (FE) Haemaphysalis flava ticks, with a special focus on the analysis of those uncharacterized proteins. Saliva was collected from PE and FE adult female H. flava ticks. Saliva proteins were analyzed by high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS-MS). MS data were searched against an in-house salivary gland transcriptome library for identification of tick-derived proteins. Abundances of proteins were compared between PE and FE ticks. The uncharacterized proteins detected in saliva were further bioinformatically analyzed. In total, 614 proteins were identified including 94 host proteins and 520 tick-derived proteins. The 226 tick-derived high-confidence proteins were classified into 10 categories: transporters, enzymes, protease inhibitors, immunity-related proteins, lipocalins, glycine-rich proteins, muscle proteins, secreted proteins, uncharacterized proteins and others. A total of 98 proteins were shared in both PE and FE with 74 only in PE and 54 only in FE. Abundances of 24 shared proteins were significantly higher in PE. The profile of top 15 most abundant proteins was also different between PE and FE ticks. The 65 uncharacterized proteins detected in tick saliva were branched into subclusters 1 A, 1B, 2, 3 A, 3B and 3 C based on particular motifs like RGD, LRR, indicating their diverse predicted functions like anti-coagulation, regulation of innate immune, or other functions. This study provides and compares saliva proteomes of H. flava ticks in two feeding stages with special cluster analysis on the uncharacterized proteins. Further investigations are needed to confirm the roles of these uncharacterized proteins in ticks.

Comparative analysis of the anticoagulant activities and immunogenicity of HSC70 and HSC70TKD of Haemaphysalis flava.

BACKGROUND: Haemaphysalis flava is a hematophagous ectoparasite that acquires the nutrition needed for development and reproduction by sucking blood and digesting the blood meal. During blood-sucking and blood-meal digestion, the prevention of blood coagulation is important for this tick. Previous studies have shown that heat shock cognate 70 (HSC70) protein has certain anticoagulant activities, but its immunogenicity remains unclear. Also, whether the mutation of individual bases of the TKD-like peptide of HSC70 through the overlap extension method can change its anticoagulant activities and immunogenicity remains to be investigated. METHODS: The gene encoding the HSC70 protein was cloned from a complementary DNA library synthesized from H. flava. The coding gene of the TKD-like peptide of HSC70 was mutated into a TKD peptide coding gene (HSC70TKD) using the overlap extension method. Escherichia coli prokaryotic expression plasmids were constructed to obtain the recombinant proteins of HSC70 (rHSC70) and HSC70TKD (rHSC70TKD). The purified rHSC70 and rHSC70TKD were evaluated at different concentrations for anticoagulant activities using four in vitro clotting assays. Emulsifying recombinant proteins with complete and incomplete Freund's adjuvants were subcutaneously immunized in Sprague Dawley rats. The serum antibody titers and serum concentrations of interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected using an indirect enzyme-linked immunosorbent assay to assess the immunogenicity of rHSC70 and rHSC70TKD. RESULTS: The open reading frame of HSC70 was successfully amplified and found to have a length of 1958 bp. The gene encoding the TKD-like peptide of HSC70 was artificially mutated, with the 1373-position adenine (A) of the original sequence mutated into guanine (G), the 1385-position cytosine (C) mutated into G and the 1386-position G mutated into C. rHSC70 and rHSC70TKD that fused with His-tag were obtained using the expression plasmids pET-28a-HSC70 and pET-28a-HSC70TKD, respectively. rHSC70 and rHSC70TKD prolonged the thrombin time (TT) and reduced the fibrinogen (FIB) content in the plasma, but did not affect the prothrombin time (PT) or activated partial thromboplastin time (APTT) when compared to the negative control. Interestingly, the ability of rHSC70TKD to prolong the TT and reduce the FIB content in the plasma was better than that of rHSC70. The specific antibody titers of both rHSC70 and rHSC70TKD in rat serum reached 1:124,000 14 days after the third immunization. The serum concentration of IFN-γ in the rHSC70TKD group was higher than that in the rHSC70 group. The rHSC70 group has the highest serum concentration of IL-4, and the serum concentration of IL-4 in the rHSC70TKD group was higher than that in the negative group. CONCLUSIONS: rHSC70 and rHSC70TKD exhibited anticoagulant activities by prolonging the TT and reducing the FIB content in vitro. rHSC70TKD had better anticoagulant activities than rHSC70. Both rHSC70 and rHSC70TKD had good immunogenicity and induced humoral and cellular immunity.

Protein profiling of hemolymph in Haemaphysalis flava ticks.

BACKGROUND: Tick hemolymph bathes internal organs, acts as an exchange medium for nutrients and cellular metabolites, and offers protection against pathogens. Hemolymph is abundant in proteins. However, there has been limited integrated protein analysis in tick hemolymph thus far. Moreover, there are difficulties in differentiating tick-derived proteins from the host source. The aim of this study was to profile the tick/host protein components in the hemolymph of Haemaphysalis flava. METHODS: Hemolymph from adult engorged H. flava females was collected by leg amputation from the Erinaceus europaeus host. Hemolymph proteins were extracted by a filter-aided sample preparation protocol, digested by trypsin, and assayed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS raw data were searched against the UniProt Erinaceidae database and H. flava protein database for host- and tick-derived protein identification. Protein abundance was further quantified by intensity-based absolute quantification (iBAQ). RESULTS: Proteins extracted from hemolymph unevenly varied in size with intense bands between 100 and 130 kDa. In total, 312 proteins were identified in the present study. Therein 40 proteins were identified to be host-derived proteins, of which 18 were high-confidence proteins. Top 10 abundant host-derived proteins included hemoglobin subunit-α and subunit-ß, albumin, serotransferrin-like, ubiquitin-like, haptoglobin, α-1-antitrypsin-like protein, histone H2B, apolipoprotein A-I, and C3-ß. In contrast, 169 were high-confidence tick-derived proteins. These proteins were classified into six categories based on reported functions in ticks, i.e., enzymes, enzyme inhibitors, transporters, immune-related proteins, muscle proteins, and heat shock proteins. The abundance of Vg, microplusin and α-2-macroglobulin was the highest among tick-derived proteins as indicated by iBAQ. CONCLUSIONS: Numerous tick- and host-derived proteins were identified in hemolymph. The protein profile of H. flava hemolymph revealed a sophisticated protein system in the physiological processes of anticoagulation, digestion of blood meal, and innate immunity. More investigations are needed to characterize tick-derived proteins in hemolymph.

Identification of a new species of Ixodes Latreille, 1795 (Acari: Ixodidae), parasite of hog badgers (Carnivora: Mustelidae) in China.

Ixodes hunanensis n. sp. (Acari: Ixodidae), is identified based on the morphological characteristics and molecular biological analyses of males and females ex hog badger, Arctonyx collaris Cuvier (Carnivora: Mustelidae) from China. Adults of this new species are similar to those of other species of the subgenus Pholeoixodes Schulze, 1942, from which they can be distinguished by the shape of basis capituli, development of cornua, size of porose areas, shape, and size of spurs on coxae and phylogenetic analyses of the cox1 and 16S rRNA sequences.

Metagenomics of the midgut microbiome of Rhipicephalus microplus from China.

BACKGROUND: Ticks, which are ectoparasites of animals, may carry multiple pathogens. The cattle tick Rhipicephalus microplus is an important bovine parasite in China. However, the midgut microbiome of R. microplus from China has not been characterized via metagenomic methods. METHODS: Rhipicephalus microplus were collected from cattle in the city of Changsha in Hunan province, China. The DNA of the midgut contents was extracted from fully engorged adult female R. microplus. A DNA library was constructed and sequenced using an Illumina HiSeq sequencing platform. SOAPdenovo software was used to assemble and analyze the clean data. The latent class analysis algorithm applied to system classification by MEGAN software was used to annotate the information on the species' sequences. DIAMOND software was used to compare unigenes with the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and functional annotation was carried out based on the results of the comparison. RESULTS: The dominant phyla in the five samples were Firmicutes, Proteobacteria, and Actinobacteria. Streptococcus, Mycobacterium, Anaplasma, Enterococcus, Shigella, Lactobacillus, Brachyspira, Pseudomonas, Enterobacter, Bacillus, and Lactococcus were the dominant genera in the five samples. The endosymbiotic bacterium Wolbachia was also detected in all of the samples. Mycobacterium malmesburyense, Streptococcus pneumoniae, Anaplasma phagocytophilum, Enterococcus faecium, Shigella sonnei, Enterococcus faecalis, Lactobacillus casei, Brachyspira hampsonii, Pseudomonas syringae, Enterobacter cloacae, and Lactococcus garvieae were the dominant species in the five samples. In addition to these bacterial species, we also detected some eukaryotes, such as Rhizophagus irregularis, Enterospora canceri, Smittium culicis, Zancudomyces culisetae, Trachipleistophora hominis, and viruses such as orf virus, human endogenous retrovirus type W, enzootic nasal tumor virus of goats, bovine retrovirus CH15, and galidia endogenous retrovirus in all of the samples at the species level. The results of the annotated KEGG pathway predictions for the gene functions of the midgut microflora of R. microplus indicated genes involved in lipid and amino acid metabolism, infectious diseases (e.g., Streptococcus pneumonia infection, human granulocytic anaplasmosis, Shigella sonnei infection, Salmonella enterica infection, and pathogenic Escherichia coli infection), and cancer. CONCLUSIONS: Our study revealed that the midgut microbiome of R. microplus is not only composed of a large number of bacteria, but that a portion also comprises eukaryotes and viruses. The data presented here enhance our understanding of this tick's midgut microbiome and provide fundamental information for the control of ticks and tick-borne diseases.

Microbiome analysis of the midguts of different developmental stages of Argas persicus in China.

Argas persicus is an ectoparasite of poultry. The bacterial community structure and the pathogenic bacteria associated with different developmental stages of A. persicus have implications for control. Argas persicus were collected from chickens in the city of Jiuquan in Gansu, China. Bacterial DNA was extracted from the midgut contents of blood engorged larvae, nymphs and adult females. The V3-V4 hypervariable regions of 16S rRNA genes were sequenced using the IonS5™XL platform. Identification of Rickettsia spp. and detection of Coxiella burnetii were performed using PCR on target genes. The bacterial diversity within larvae was the highest and the bacterial diversity within nymphs was greater than that of adults. At different classification levels, seven bacterial phyla were common phyla, 27 genera were common genera, and 18 species were common species in the three samples. At the phylum level, Proteobacteria showed a marked predominance in all samples. Rickettsia, Stenotrophomonas, Spiroplasma, and Coxiella were the dominant bacteria at the genus level. The Rickettsia species in A. persicus was identified as Rickettsia hoogstraalii and the Coxiella species was identified as a Coxiella-like endosymbiont. Additionally, some bacterial species such as Pseudomonas geniculata, Sphingomonas koreensis, and Acinetobacter haemolyticus were reported here for the first time in A. persicus.

Cloning, expression, and function of ferritins in the tick Haemaphysalis flava.

The full-length cDNA of two ferritins of Haemaphysalis flava were cloned after which recombinant Hf-FER1 and Hf-FER2 were expressed and their function was analyzed. In addition, RNA interference (RNAi) based on the injection of Hf-fer1 or Hf-fer2 dsRNA into fully engorged female ticks was performed. The cDNA encoding Hf-FER1 is 834 bp in length. It contains an iron-responsive element in the 5' untranslated region and encodes 174 amino acid residues. The full-length cDNA of Hf-FER2 contains 696 bp and encodes 199 amino acids, including a putative signal peptide sequence. Hf-FER1 and Hf-FER2 both have the ferroxidase iron center and the ferrihydrite nucleation center. The evolutionary relationship of Hf-FER1 and Hf-FER2 was established, and the predicted quaternary structures were assembled as typical spherical shells composed of 24 subunits which was demonstrated by nature PAGE. Real-time PCR showed that Hf-fer1 and Hf-fer2 were expressed in all developmental stages, with the highest expression in fully engorged females. The expression of Hf-fer1 and Hf-fer2 were relatively high in unfed larvae. Hf-fer1 was expressed in all tissues and was especially abundant in the salivary glands of fully engorged females. In contrast, the highest levels of Hf-fer2 were found in the midgut of fully engorged females, and no expression was found in the salivary glands of this life stage. Both recombinant Hf-FER1 and Hf-FER2 had iron-binding capabilities. Silencing of both Hf-fer1 and Hf-fer2 affected fecundity. Compared to the control, the percentage of ticks that laid eggs in the Hf-fer1 and Hf-fer2 RNAi groups was 73.3% and 66.7%, respectively. The silenced ticks that laid eggs had lower egg weight to body weight ratios, and the eggs had abnormal morphologies. The hatchability of eggs with normal morphology in the Hf-fer1 and Hf-fer2 silenced groups was 47.8% and 22.8%, respectively, which was significantly different from the control group (P < 0.005). These findings indicate that Hf-FER1 and Hf-FER2 play important roles in the iron storage of H. flava.

Characterization of AV422 from Haemaphysalis flava ticks in vitro.

Ticks are hematophagous ectoparasites and cause a major public health threat worldwide. Development of anti-tick vaccines is regarded to be an optimal alternative for tick control. AV422, a unique protein in ticks, is secreted into hosts during blood-feeding, but its roles are not confirmed in Haemaphysalis flava ticks. We retrieved a gene fragment encoding AV422 from a transcriptome dataset of H. flava, and based on it, we reconstructed the full length of AV422 from H. flava (Hf-AV422) by rapid amplification of cDNA ends. Expression profiles of Hf-AV422 in whole ticks and organs of different engorgement levels were determined by qPCR. Then its opening reading frame (ORF) was expressed in Escherichia coli strain BL21 (DE3). The prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT) assays were conducted to test anticoagulant activities of the purified recombinant protein (rHf-AV422). The full length of AV422 was 1152 bp. Hf-AV422 showed to be conserved as indicated by multiple sequence alignment. Expression of Hf-AV422 was significantly higher in salivary glands and cuticles than in ovaries. Its expression in whole ticks decreased during engorgement with the highest levels in 1/4 engorged ticks. rHf-AV422 prolonged PT, APTT and TT when incubated with rabbit plasma. Our data demonstrated that Hf-AV422 is a conserved salivary protein with anticoagulant activity. Further studies are needed to test in detail its functional properties to ensure it an adequate antigen candidate for the development of broad-spectrum vaccines against ticks.

Variation of mitochondrial minichromosome composition in Hoplopleura lice (Phthiraptera: Hoplopleuridae) from rats.

BACKGROUND: The family Hoplopleuridae contains at least 183 species of blood-sucking lice, which widely parasitize both mice and rats. Fragmented mitochondrial (mt) genomes have been reported in two rat lice (Hoplopleura kitti and H. akanezumi) from this family, but some minichromosomes were unidentified in their mt genomes. METHODS: We sequenced the mt genome of the rat louse Hoplopleura sp. with an Illumina platform and compared its mt genome organization with H. kitti and H. akanezumi. RESULTS: Fragmented mt genome of the rat louse Hoplopleura sp. contains 37 genes which are on 12 circular mt minichromosomes. Each mt minichromosome is 1.8-2.7 kb long and contains 1-5 genes and one large non-coding region. The gene content and arrangement of mt minichromosomes of Hoplopleura sp. (n = 3) and H. kitti (n = 3) are different from those in H. akanezumi (n = 3). Phylogenetic analyses based on the deduced amino acid sequences of the eight protein-coding genes showed that the Hoplopleura sp. was more closely related to H. akanezumi than to H. kitti, and then they formed a monophyletic group. CONCLUSIONS: Comparison among the three rat lice revealed variation in the composition of mt minichromosomes within the genus Hoplopleura. Hoplopleura sp. is the first species from the family Hoplopleuridae for which a complete fragmented mt genome has been sequenced. The new data provide useful genetic markers for studying the population genetics, molecular systematics and phylogenetics of blood-sucking lice.

Microbiome analysis of the saliva and midgut from partially or fully engorged female adult Dermacentor silvarum ticks in China.

Dermacentor silvarum is widely distributed in northern China and transmits several pathogens that cause diseases in humans and domestic animals. We analysed the comprehensive bacterial community of the saliva and midgut from partially and fully engorged female adult D. silvarum. Dermacentor silvarum samples were collected from Guyuan, China. Bacterial DNA was extracted from the saliva and midgut contents of partially or fully engorged female adult D. silvarum. Sequencing of the V3-V4 hypervariable regions of the 16S rRNA genes was performed using the IonS5TMXL platform. The bacterial diversity in saliva was higher than in the midgut. The bacterial diversity of saliva from fully engorged ticks was greater than in partially engorged tick saliva. The bacterial diversity in midguts from partially engorged ticks was greater than in fully engorged tick midguts. Proteobacteria was the most dominant bacterial phylum in all of the samples. Twenty-nine bacterial genera were detected in all of the samples. Rickettsia, Anaplasma, and Stenotrophomonas were the main genera. The symbionts Coxiella, Arsenophonus, and Wolbachia were also detected in all of the samples. Eight bacterial species were identified in all of the experimental samples. Anaplasma marginale was reported for the first time in D. silvarum.

Comparative analyses of the mitochondrial genome of the sheep ked Melophagus ovinus (Diptera: Hippoboscidae) from different geographical origins in China.

The sheep ked Melophagus ovinus is mainly found in Europe, Northwestern Africa, and Asia. Although M. ovinus is an important ectoparasite of sheep in many countries, the population genetics, molecular biology, and systematics of this ectoparasite remain poorly understood. Herein, we determined the mitochondrial (mt) genome of M. ovinus from Gansu Province, China (MOG) and compared with that of M. ovinus Xinjiang Uygur Autonomous Region, China (MOX). The mt genome sequence (15,044 bp) of M. ovinus MOG was significantly shorter (529 bp) than M. ovinus MOX. Nucleotide sequence difference in the whole mt genome except for non-coding region was 0.37% between M. ovinus MOG and MOX. For the 13 protein-coding genes, comparison revealed sequence divergences at both the nucleotide (0-1.1%) and amino acid (0-0.59%) levels between M. ovinus MOG and MOX, respectively. Interestingly, the cox1 gene of M. ovinus MOX is predicted to employ unusual mt start codons AAA, which has not been predicted previously for any parasite genome. Phylogenetic analyses showed that M. ovinus (Hippoboscoidea) is related to the superfamilies Oestroidea + Muscoidea. Our results have also indicated the paraphylies of the four families (Anthomyiidae, Calliphoridae, Muscidae, and Oestridae) and two superfamilies (Oestroidea and Muscoidea). This mt genome of M. ovinus provides useful molecular markers for studies into the population genetics, molecular biology, and systematics of this ectoparasite.

Microbial population analysis of the midgut of Melophagus ovinus via high-throughput sequencing.

BACKGROUND: Melophagus ovinus, one of the most common haematophagous ectoparasites of sheep, can cause anaemia and reductions in weight gain, wool growth and hide value. However, no information is available about the microfloral structure of the midgut of this ectoparasite. In the present study, we investigated the microbial community structure of the midgut contents of fully engorged female and male M. ovinus using Illumina HiSeq. RESULTS: The phylum showing the highest abundance was Proteobacteria (99.9%). The dominant bacterial genera in females and males were Bartonella, Arsenophonus and Wolbachia. Some less abundant bacterial genera were also detected, including Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus. CONCLUSIONS: Bartonella, Arsenophonus and Wolbachia were the dominant bacterial genera in the midgut of female and male M. ovinus. Although detected, Enterobacter, Acinetobacter, Halomonas, Shewanella, Bacillus and Staphylococcus showed low abundances. Importantly, this is the first report of the presence of Arsenophonus, Wolbachia, Enterobacter, Halomonas, Shewanella, Bacillus and Staphylococcus in the midgut of M. ovinus.