Acaricidal activity of synthesized titanium dioxide nanoparticles using Calotropis gigantea against Rhipicephalus microplus and Haemaphysalis bispinosa

Objective:To assess the acaricidal activity of titanium dioxide nanoparticles(TiO_2 NPs)synthesized from flower aqueous extract of Calotropis gigantea(C.gigantea)against the larvae of Rhipicephalus(Boophilus)microplus[R.(B.)microplus]and the adult of Haemaphrysalis bispinosa(H.bispinosa).Methods:The lyophilized C.gigantea flower aqueous extract of 50 mg was added with 100 mL of TiO(OH_2)(10 mM)and magnetically stirred for 6 h.Synthesized TiO_2 NPs were characterized by X-ray diffraction(XRD).Fourier transform infrared spectroscopy(FTIR),Scanning electron microscopy(SEM),and Energy dispersive X-ray spectroscopy(EDX).The synthesised TiO_2 NPs were tested against the larvae of R(B.)microplus and adult of H.bispinosa were exposed to filter paper impregnated method.Results:XRD confirmed the crystalline nature of the nanoparticles with the mean size of 10.52 nm.The functional groups for synthesized TiO_2NPs were 1405.19,and 1053.45 cm^(-1)for-NH_2 bending,primary amines and amides and 1053.84and 1078.45 cm^(-1)for C-O.SEM micrographs of the synthesized TiO_2 NPs showed the aggregated and spherical in shape.The maximum efficacy was observed in the aqueous flower extract of C.gigantea and synthesized TiO_2 NPs against R.(B.)microplus(LC_(50)=24.63 and 5.43 mg/L and r^2=0.960 and 0.988)and against H.bispinosa(LC_(50)=35.22 and 9.15 mg/L and r^2=0.969 and 0.969).respectively.Conclusions:The synthesized TiO_2 NPs were highly stable and had significant acaricidal activity against the larvae of R.(B.)microplus and adult of H.bispinosa.This study provides the first report of synthesized TiO_2 NPs and possessed excellent anti-parasitic activity.

Construction of Larval cDNA Expression Library and Immunological Identification of Positive Clones in Tick Haemaphysalis qinghaiensis

A primary cDNA expression library with a titer of 5.0 × 105 PFU mL-1 was constructed from mRNA extracted from larval Haemaphysalis qinghaiensis ticks in order to identify certain genes,which would then be used as candidate molecules for development of effective vaccines to control this parasite.Totally 11 positive clones,which designated as HqL01-11,were obtained by immunoscreening of the library using a polyclonal antibody generated in rabbit with larval tick protein extract.Results of sequence analysis from BLASTN searching revealed that 6 of them had no significant homology with the adult H.qinghaiensis ticks’ known genes,4 of them had no significant homology with all genes deposited in GenBank database.HqL07,HqL08,HqL09,and HqL11 were deposited to GenBank database,and accession numbers were EF605263,EF605264,EF605265,and EF605266,respectively.Subsequently,HqL07 and HqL09 were expressed in vitro and the molecular weights of the corresponding expressed products were 60 and 70 kDa,respectively.Western blot analyses showed that HqL07 and HqL09 had immunogenicity.This study laid the foundation for future production of genetically engineered vaccines for the immunological control of H.qinghaiensis.

Viromes of Haemaphysalis longicornis reveal different viral abundance and diversity in free and engorged ticks

Haemaphysalis longicornis ticks,commonly found in East Asia,can transmit various pathogenic viruses,includingthe severe fever with thrombocytopenia syndrome virus(SFTSV)that has caused febrile diseases among humansin Hubei Province.However,understanding of the viromes of H.longicornis was limited,and the prevalence ofviruses among H.longicornis ticks in Hubei was not well clarified.This study investigates the viromes of bothengorged(fed)and free(unfed)H.longicornis ticks across three mountainous regions in Hubei Province from 2019to 2020.RNA-sequencing analysis identified viral sequences that were related to 39 reference viruses belonging tounclassified viruses and seven RNA viral families,namely Chuviridae,Nairoviridae,Orthomyxoviridae,Parvoviridae,Phenuiviridae,Rhabdoviridae,and Totiviridae.Viral abundance and diversity in these ticks were analysed,andphylogenetic characteristics of the Henan tick virus(HNTV),Dabieshan tick virus(DBSTV),Okutama tick virus(OKTV),and Jingmen tick virus(JMTV)were elucidated based on their full genomic sequences.Prevalenceanalysis demonstrated that DBSTV was the most common virus found in individual H.longicornis ticks(12.59%),followed by HNTV(0.35%),whereas JMTV and OKTV were not detected.These results improve our understanding of H.longicornis tick viromes in central China and highlight the role of tick feeding status and geographyin shaping the viral community.The findings of new viral strains and their potential impact on public health raisethe need to strengthen surveillance efforts for comprehensively assessing their spillover potentials.

Epidemiology of toxoplasmosis: role of the tick Haemaphysalis longicornis

Background:Toxoplasma gondii infection is mainly caused by ingestion of water or food that is contaminated with oocysts excreted by cats,or by eating raw meat containing T.gondii tissue cysts.However,oral transmission does not explain the common occurrence of toxoplasmosis in a variety of hosts,such as herbivorous animals,birds,and wild rodents.Little information exists on the maintenance of T.gondii parasites in nature and routes of transmission to domestic and wild animal hosts.Therefore,this study evaluated the role of Haemaphysalis longicornis ticks in the epidemiology of toxoplasmosis.Methods:The real-time polymerase chain reaction(qPCR)technique was used to detect the presence of T.gondii DNA in ticks collected from the field.To observe the amount of dynamic changes of T.gondii in the tick’s body and its infectivity,microinjection of green fluorescence parasites was performed.Under laboratory conditions,we evaluated if H.longicornis ticks were infected with T.gondii and their potential to transmit the infection to other hosts using traditional parasitological methods coupled with molecular detection techniques.Results:The infection rates of T.gondii parasites among field-collected adult and nymph H.longicornis ticks were 11.26%and 5.95%,respectively.T.gondii can survive and remain infective in a tick’s body for at least 15 days.We found that blood feeding of infected ticks did not transmit T.gondii to hosts,however,ingestion of infected ticks may be a transmission route between ticks and other common hosts.Conclusion:The T.gondii infection in ticks could serve as a reservoir for toxoplasmosis transmission.

Identification of tick-borne pathogen diversity by metagenomic analysis in Haemaphysalis longicornis from Xinyang,China

Background:A wide variety of pathogens could be maintained and transmitted by Haemaphysalis longicornis.The aim of this study is to systematically examine the variety of pathogens carried by Haemaphysalis longicornis,an importnatn vector,in tick-borne diseases epidemic area,and to estimate the risk of human infection imposed by tick bites.Methods:Adult questing ticks were collected in Xinyang,central China.Genomic DNA and RNA were extracted from 144 H.longicornis ticks individually,and sequenced respectively as the templates for high-throughput sequencing.Clean reads were compared against the database of NCBI nucleotide collection and specific PCR was performed to confirm the presence of pathogen.Phylogenetic analysis was performed to explore the evolutionary status of pathogens.Results:The assignment of reads to taxa based on BLASTN results revealed the existence of several potential pathogens,including Anaplasma spp.,Rickettsia spp.,Babesia sp.,as well as severe fever with thrombocytopenia syndrome bunyavirus(SFTSV).Comfirmantory PCR assays revealed the existence of Anaplasma bovis(13/144,9.03%),Anaplasma centrale(2/144,1.39%),Rickettsia heilongjiangensis(3/144,2.08%),Rickettsia sp.LON-13(1/144,0.69%),Rickettsia raoultii(5/144,3.47%),Babesia sp.(1/144,0.69%).SFTSV accounted for the highest detected pathogen with a positive rate of 18.75%(27/144).Three of the ticks(2.08%)were co-infected with SFTSV and A.bovis.Conclusion:Our study provided a broadened list of microorganism that harbored by H.longicornis.In previously unrecognized endemic regions,prokaryotic and eukaryotic infection including Anaplasma spp.,Rickettsiae spp.,and Babesia spp.should be considered,along with the well-known SFTSV for patients with tick bites history.A novel Babesia species was identified in local natural foci,which needs further investigation in the future.

An inhibitor selective for collagen-stimulated platelet aggregation from the salivary glands of hard tick Haemaphysalis longicornis and its mechanism of action

Soluble materials of salivary glands from Haemaphysalis longicornis were found to inhibit collagen, ADP, and thrombin- stimulated platelet aggregation. One inhibitory component was purified to salivary gland homogeneity by a combination of gel filtration, ion-exchange, and C8 reverse phase HPLC. The purified activity, named longicomin, is a protein of molecular weight 16 000 on SDS-PAGE under both reduced and nonreduced conditions. Collagen-mediated aggregation of platelets in plasma and of washed platelets (IC50 was approximately 60 nmol/L) was inhibited with the same efficacy. No inhibition of aggregation stimulated by other effectors, including ADP, arachidonic acid, thrombin, risto-cetin, calcium ionophore A23187, thromboxane A2 mimetic U46619 and 12-0-phorbol-13-myristate acetate, was observed . Longiconin had no effect on platelet adhension to collagen. Not only platelet aggregation but also release reaction, and increase of intracellar Ca2+ level of platelets in response to collagen were completely eliminated by longicomin . Increasing amounts of collagen are able to overcome the inhibition of aggregation by longicomin, indicating that longicomin probably shares with collagen a common receptor. In addition, collagen fibers did not emit fluorescence after incubation with isothocyanate-conjugated longicomin, indicating that longicomin did not bind directly to collagen fibers. The identification and isolation of longicomin demonstrates the existence of a new type of platelet inhibitor that should be useful to better understand the mechanism of collagen stimulation of platelets.