Plasma-Enabled Ligand Removal for Improved Catalysis: Furfural Conversion on Pd/SiO2.

A nonthermal, atmospheric He/O2 plasma (NTAP) successfully removed polyvinylpyrrolidone (PVP) from Pd cubic nanoparticles supported on SiO2 quickly and controllably. Transmission electron microscopy (TEM) revealed that the shape and size of Pd nanoparticles remain intact during plasma treatment, unlike mild calcination, which causes sintering and polycrystallinity. Using Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS), we demonstrate the quantitative estimation of the PVP plasma removal rate and control of the nanoparticle synthesis. First-principles calculations of the XPS and CO FTIR spectra elucidate electron transfer from the ligand to the metal and allow for estimates of ligand coverages. Reactivity testing indicated that PVP surface crowding inhibits furfural conversion but does not alter furfural selectivity. Overall, the data demonstrate NTAP as a more efficient method than traditional calcination for organic ligand removal in nanoparticle synthesis.

Assessment of Tick-Borne Diseases in Hainan Province, China.

China's six tropical regions include Guangdong Province, Yunnan Province, Hainan Province, Hong Kong Special Administrative Region (SAR), Macau SAR, and Taiwan, China. Hainan, seated in the southernmost tropical region of China, is home to ticks that remain active throughout all four seasons. This heightens their potential to transmit tick-borne diseases to both animals and humans. This study provides a succinct overview of the prevailing tick species' spatial distribution and offers an outline of the range and dispersion of emerging tick-borne infections in tick vectors, animal hosts, and human populations within Hainan, China.

Microwave-assisted, performance-advantaged electrification of propane dehydrogenation.

Nonoxidative propane dehydrogenation (PDH) produces on-site propylene for value-added chemicals. While commercial, its modest selectivity and catalyst deactivation hamper the process efficiency and limit operation to lower temperatures. We demonstrate PDH in a microwave (MW)-heated reactor over PtSn/SiO2 catalyst pellets loaded in a SiC monolith acting as MW susceptor and a heat distributor while ensuring comparable conditions with conventional reactors. Time-on-stream experiments show active and stable operation at 500°C without hydrogen addition. Upon increasing temperature or feed partial pressure at high space velocity, catalysts under MWs show resistance in coking and sintering, high activity, and selectivity, starkly contrasting conventional reactors whose catalyst undergoes deactivation. Mechanistic differences in coke formation are exposed. Gas-solid temperature gradients are computationally investigated, and nanoscale temperature inhomogeneities are proposed to rationalize the different performances of the heating modes. The approach highlights the great potential of electrification of endothermic catalytic reactions.

Response of Paleogene Fine-Grained Clastic Rock Deposits in the South Qiangtang Basin to Environments and Thermal Events on the Qinghai-Tibet Plateau.

The Chamdo Basin is a secondary basin in the eastern part of Tibet China and is one of the most promising of petroliferous basins for new petroleum exploration. The Qamdo Basin records a complex burial history from the Mesozoic to the Cenozoic; however, the poorly constrained sedimentology of Cenozoic strata in this basin has severely obscured the overall profile and impeded further explorations of oil and gas resources. Here, we conduct whole-rock geochemical analyses of major, trace, and rare earth elements in fine-grained clastic rocks of the Paleocene Gongjue Formation, Qamdo Basin to reveal depositional environments, provenance, and tectonic setting. Petrologically, the Gongjue Formation is dominated by red fine-grained sandy mudstones/siltstones with ripple marks. The high values of the chemical index of alteration (avg. of 78.93), chemical index of weathering (avg. of 90.10), and index of compositional variability (avg. of 2.5) suggest that the basin has undergone heavy weathering. Cross-plots of La vs Th, Th vs Sc vs Zr/10, and Th vs Co vs Zr/10 reveal a continental arc tectonic setting. Paleosalinity (Sr/Ba), paleoclimate (Sr/Cu), and redox proxies (V/Cr, U/Th, and enrichment factors of Mo and U) indicate brackish to saline and oxidizing paleowater masses during deposition of the Gongjue Formation. Provenance analyses via elements and petrology reveal that sediments in the Gongjue Formation are mainly derived from intermediate-acidic rocks of the upper crust. We conclude that the first and third members are more arid climate and heavily chemically weathered than the second member. In combination with previous studies of the structural evolution of the Qamdo Basin since the Paleogene, a model is built to describe the sedimentary environment and evolution of the Qamdo Basin during transition to the Paleocene. The first and third members, i.e., the Eg1 and Eg3 members of the Gongjue Formation, are dominated by an oxidizing environment of seawater-saltwater, and the climate ranges from warm and humid to arid and hot, with relatively stable environmental changes. The Eg2 member of the Gongjue Formation is dominated by an oxidizing environment of seawater-saltwater, and the climate ranges from warm and humid to arid and hot, with more frequent environmental evolution. Our model aids in better understanding of the Paleocene climate evolution of the eastern Tibetan Plateau.

Enhanced Catalytic Hydrodeoxygenation of Activated Carbon-Supported Metal Catalysts via Rapid Plasma Surface Functionalization.

We employ a nonthermal, He/O2 atmospheric plasma as an efficient surface functionalization method of activated carbons. We show that plasma treatment rapidly increases the surface oxygen content from 4.1 to 23.4% on a polymer-based spherical activated carbon in 10 min. Plasma treatment is 3 orders of magnitude faster than acidic oxidation and introduces a diverse range of carbonyl (C═O) and carboxyl (O-C═O) functionalities that were not found with acidic oxidation. The increased oxygen functionalities reduce the particle size of a high 20 wt % loading Cu catalyst by >44% and suppress the formation of large agglomerates. Increased metal dispersion exposes additional active sites and improves the yield of hydrodeoxygenation of 5-hydroxymethyl furfural to 2,5-dimethyl furan, an essential compound for biofuel replacement, by 47%. Surface functionalization via plasma can advance catalysis synthesis while being rapid and sustainable.

Identification and Characterization of Rhipicephalus microplus ATAQ Homolog from Haemaphysalis longicornis Ticks and Its Immunogenic Potential as an Anti-Tick Vaccine Candidate Molecule.

Although vaccines are one of the environmentally friendly means to prevent the spread of ticks, there is currently no commercial vaccine effective against Haemaphysalis longicornis ticks. In this study, we identified, characterized, localized, and evaluated the expression patterns, and tested the immunogenic potential of a homologue of Rhipicephalus microplus ATAQ in H. longicornis (HlATAQ). HlATAQ was identified as a 654 amino acid-long protein present throughout the midgut and in Malpighian tubule cells and containing six full and one partial EGF-like domains. HlATAQ was genetically distant (homology < 50%) from previously reported ATAQ proteins and was expressed throughout tick life stages. Its expression steadily increased (p < 0.001) during feeding, reached a peak, and then decreased slightly with engorgement. Silencing of HlATAQ did not result in a phenotype that was significantly different from the control ticks. However, H. longicornis female ticks fed on a rabbit immunized with recombinant HlATAQ showed significantly longer blood-feeding periods, higher body weight at engorgement, higher egg mass, and longer pre-oviposition and egg hatching periods than control ticks. These findings indicate that the ATAQ protein plays a role in the blood-feeding-related physiological processes in the midgut and Malpighian tubules and antibodies directed against it may affect these tissues and disrupt tick engorgement and oviposition.

Tuning the reactivity of carbon surfaces with oxygen-containing functional groups.

Oxygen-containing carbons are promising supports and metal-free catalysts for many reactions. However, distinguishing the role of various oxygen functional groups and quantifying and tuning each functionality is still difficult. Here we investigate the role of Brønsted acidic oxygen-containing functional groups by synthesizing a diverse library of materials. By combining acid-catalyzed elimination probe chemistry, comprehensive surface characterizations, 15N isotopically labeled acetonitrile adsorption coupled with magic-angle spinning nuclear magnetic resonance, machine learning, and density-functional theory calculations, we demonstrate that phenolic is the main acid site in gas-phase chemistries and unexpectedly carboxylic groups are much less acidic than phenolic groups in the graphitized mesoporous carbon due to electron density delocalization induced by the aromatic rings of graphitic carbon. The methodology can identify acidic sites in oxygenated carbon materials in solid acid catalyst-driven chemistry.

Selective hydrogenation via precise hydrogen bond interactions on catalytic scaffolds.

The active site environment in enzymes has been known to affect catalyst performance through weak interactions with a substrate, but precise synthetic control of enzyme inspired heterogeneous catalysts remains challenging. Here, we synthesize hyper-crosslinked porous polymer (HCPs) with solely -OH or -CH3 groups on the polymer scaffold to tune the environment of active sites. Reaction rate measurements, spectroscopic techniques, along with DFT calculations show that HCP-OH catalysts enhance the hydrogenation rate of H-acceptor substrates containing carbonyl groups whereas hydrophobic HCP- CH3 ones promote non-H bond substrate activation. The functional groups go beyond enhancing substrate adsorption to partially activate the C = O bond and tune the catalytic sites. They also expose selectivity control in the hydrogenation of multifunctional substrates through preferential substrate functional group adsorption. The proposed synthetic strategy opens a new class of porous polymers for selective catalysis.

Detection and phylogenetic analysis of tick-borne bacterial and protozoan pathogens in a forest province of eastern China.

Ticks, as obligate blood-sucking ectoparasites, feed on a broad range of vertebrates and transmit a great diversity of pathogenic microorganisms. Some tick-borne pathogens (TBPs) are endemic in China, whereas epidemiological studies are limited in Jiangxi, a forest province located in eastern China. Here, we have determined the positivity rates of TBPs in humans, rodents, dogs, goats and ticks, and performed the molecular characterization of TBPs in Jiangxi province. We found a high positivity rate of TBPs in the collected samples, demonstrating 23 (12.92%) samples positive for more than one TBPs. Of those, 11 (6.18%) samples were positive for Rickettsia spp., six (3.37%) Ehrlichia spp./Anaplasma spp., one (0.56%) Bartonella spp., two (1.12%) Borrelia spp., and five (2.81%) Babesia spp. The positivity rates of TBPs varied among ticks, animals, and humans as follow: goats (14/37, 37.84%), ticks (8/35, 22.86%), and dogs (1/11, 9.09%). Humans and rodents were negative for TBP presence. Phylogenetic analyses of these TBP sequences revealed the presence of Rickettsia japonica, Ehrlichia minasensis, and an unclassified Babesia spp. in goats, and Anaplasma phagocytophilum, Borrelia valaisiana, and an unclassified Bartonella spp. in ticks. Furthermore, R. japonica infection was exclusively found in goats with the positivity rate of 29.73%. Our study is the first report of R. japonica in goats around the world. These findings suggest high TBP positivity rates among goats, ticks, and dogs, and diverse TBPs in goats and ticks in the studied sites. Therefore, our results underscore the urgent need to assess TBP-tick-vertebrate-environment interactions and the risk of tick borne disease exposure in humans in the future.

Haemaphysalis longicornis calreticulin is not an effective molecular tool for tick bite diagnosis and disruption of tick infestations.

BACKGROUND: Tick calreticulin (CRT) is a calcium-binding protein secreted into the host during blood feeding. It has been used as a biomarker of tick exposure and has potential as an anti-tick vaccine, but there is no information about these uses for Haemaphysalis longicornis CRT (HlCRT). We synthesized recombinant H. longicornis CRT (rHlCRT) and evaluated its potential for tick bite diagnosis and for disrupting tick infestations. METHODS: The responses of mice and rabbits exposed to H. longicornis ticks were measured with ELISA to determine the antibody level against rHlCRT. To evaluate the effects of rHlCRT-induced anti-tick immunity, engorgement weight, tick engorgement index (TEI), feeding duration, ecdysis rate, and egg weight per engorged tick were compared between ticks fed on immunized and normal mice. RESULTS: Mean anti-tick CRT antibody levels in sera collected from mice at 1 and 15 days after primary tick exposure were not significantly different from the mean antibody levels in negative control mice that were not bitten by ticks (all P values > 0.05). No significant anti-HlCRT IgG responses developed in mice after second exposure to tick bites compared with the level of anti-HlCRT antibody response in negative control mice (all P values > 0.25). For rabbits, no significant differences in the antibody levels were observed in animals before challenge infestation and after tick exposures, and in animals after two tick exposures (all P values > 0.10). There were no significant differences in the body weight of ticks fed on immunized and normal mice (all P values > 0.15). No significant differences in TEI were observed between ticks fed on immunized mice and normal control mice (all P values > 0.50). There were no significant differences in feeding duration for female ticks, and feeding duration and ecdysis rate for nymphs in the experimental and control groups (all P values > 0.10 for feeding duration and P value = 0.19 for ecdysis rate). We did not observe a significant difference in egg weight per tick in the rHlCRT-immunized and the control groups (P = 0.88). CONCLUSIONS: HlCRT in H. longicornis tick saliva proteins appears to be nonimmunogenic to mammalian hosts like mice and rabbits. Vaccination with rHlCRT did not generate effective immunity against parthenogenetic and bisexual H. longicornis nymphs or female ticks. These results indicate that HlCRT is not a suitable molecular candidate for H. longicornis tick bite diagnosis and not effective for the disruption of tick infestations.

Severe Rhipicephalus sanguineus tick infestation in a home from rural area of Nanchang in China.

On October 13, 2021, a tick infestation occurred in a home in rural area of Nanchang city, China, and we were asked to inspect the tick infestation. Ticks were collected in the largest number on courtyard door jambs, followed by living room and bedroom door jambs. Ticks were identified morphologically as Rhipicephalus sanguineus adults. The 16S rRNA analysis effectively distinguished the ticks in this study from other Rhipicephalus species, including R. sanguineus south-east, temperate and tropical lineages and identified genetically as R. sanguineus south China lineage. Tick samples were subjected to conventional PCR analysis and detected negative for the presence of tick-borne pathogens. Our findings indicate that there was low transmission risk of tick-borne pathogens to humans in the tick-infested home. Further studies are needed to proactively investigate the tick species in Nanchang, and determine the presence of tick-borne pathogens for assessing their threat to human health in the region.

In Situ Tracking of Nonthermal Plasma Etching of ZIF-8 Films.

Surface characterization is critical for understanding the processes used for preparing catalysts, sorbents, and membranes. Nonthermal plasma (NTP) is a process that achieves high reactivity at low temperatures and is used to tailor the surface properties of materials. In this work, we combine the capabilities of infrared reflection absorption spectroscopy (IRRAS) with NTP for the in situ interrogation of zeolitic imidazolate framework-8 (ZIF-8) thin films to probe modifications in the material induced by oxygen and nitrogen plasmas. The IRRAS measurements in oxygen plasma reveal etching of organic ligands with sequential removal of the methyl group and imidazole ring and with the formation of carbonyl moieties (C═O). In contrast, nitrogen plasma induces mild etching and grafting of nitrile groups (-C≡N). Scanning electron microscopy imaging shows that oxygen plasma, at prolonged times, significantly degrades the ZIF-8 film at the grain boundaries. Treatment of ZIF-8 membranes using mild plasma conditions yields a fivefold enhancement for H2/N2 and CO2/CH4 ideal selectivities and an eightfold enhancement for CO2/N2 ideal selectivity. Additionally, the new tools described here can be used for spectroscopic in situ tracking of plasma-induced chemistry on thin films in general.

Polyethylene Hydrogenolysis at Mild Conditions over Ruthenium on Tungstated Zirconia.

Plastics waste has become a major environmental threat, with polyethylene being one of the most produced and hardest to recycle plastics. Hydrogenolysis is potentially the most viable catalytic technology for recycling. Ruthenium (Ru) is one of the most active hydrogenolysis catalysts but yields too much methane. Here we introduce ruthenium supported on tungstated zirconia (Ru-WZr) for hydrogenolysis of low-density polyethylene (LDPE). We show that the Ru-WZr catalysts suppress methane formation and produce a product distribution in the diesel and wax/lubricant base-oil range unattainable by Ru-Zr and other Ru-supported catalysts. Importantly, the enhanced performance is showcased for real-world, single-use LDPE consumables. Reactivity studies combined with characterization and density functional theory calculations reveal that highly dispersed (WO x )n clusters store H as surface hydroxyls by spillover. We correlate this hydrogen storage mechanism with hydrogenation and desorption of long alkyl intermediates that would otherwise undergo further C-C scission to produce methane.

Spatiotemporal Dynamics of Scrub Typhus in Jiangxi Province, China, from 2006 to 2018.

Background: Scrub typhus (ST) has become a significant potential threat to public health in Jiangxi. Further investigation is essential for the control and management of the spatiotemporal patterns of the disease. Methods: Time-series analyses, spatial distribution analyses, spatial autocorrelation analysis, and space-time scan statistics were performed to detect spatiotemporal dynamics distribution of the incidence of ST. Results: From 2006 to 2018, a total of 5508 ST cases occurred in Jiangxi, covering 79 counties. The number of ST cases increased continuously from 2006 to 2018, and there was obvious seasonality during the variation process in each year, with a primary peak in autumn (September to October) and a smaller peak in summer (June to August). From 2007 to 2018, the spatial distribution of the ST epidemic was significant heterogeneity, and Nanfeng, Huichang, Xunwu, Anyuan, Longnan, and Xinfeng were hotspots. Seven spatiotemporal clusters were observed using Kulldorff's space-time scan statistic, and the most likely cluster only included one county, Nanfeng county. The high-risk areas of the disease were in the mountainous, hilly region of Wuyi and the southern mountainous region of Jiangxi. Conclusions: Targeted interventions should be executed in high-risk regions for the precise prevention and control of ST.

PLK:Δgra9 Live Attenuated Strain Induces Protective Immunity Against Acute and Chronic Toxoplasmosis.

Toxoplasmosis is a zoonotic parasitic disease caused by the obligate intracellular protozoa Toxoplasma gondii, which threatens a range of warm-blooded mammals including humans. To date, it remains a challenge to find safe and effective drug treatment or vaccine against toxoplasmosis. In this study, our results found that the development of a mutant strain based on gene disruption of dense granule protein 9 (gra9) in type II PLK strain decreased parasite replication in vivo, severely attenuated virulence in mice, and significantly reduced the formation of cysts in animals. Hence, we developed an immunization scheme to evaluate the protective immunity of the attenuated strain of Δgra9 in type II PLK parasite as a live attenuated vaccine against toxoplasmosis in the mouse model. Δgra9 vaccination-induced full immune responses characterized by significantly high levels of pro-inflammatory cytokine interferon gamma (IFN-γ) and interleukin-12 (IL-12), maintained the high T. gondii-specific immunoglobulin G (IgG) level, and mixed high IgG1/IgG2a levels. Their levels provided the complete protective immunity which is a combination of cellular and humoral immunity in mouse models against further infections of lethal doses of type I RH, type II PLK wild-type tachyzoites, or type II PLK cysts. Results showed that Δgra9 vaccination proved its immunogenicity and potency conferring 100% protection against acute and chronic T. gondii challenges. Together, Δgra9 vaccination provided safe and efficient immune protection against challenging parasites, suggesting that PLK:Δgra9 is a potentially promising live attenuated vaccine candidate.

Subolesin vaccination inhibits blood feeding and reproduction of Haemaphysalis longicornis in rabbits.

BACKGROUND: Ticks can transmit numerous tick-borne pathogens and cause a huge economic loss to the livestock industry. Tick vaccines can contribute to the prevention of tick-borne diseases by inhibiting tick infestation or reproduction. Subolesin is an antigenic molecule proven to be a potential tick vaccine against different tick species and even some tick-borne pathogens. However, its effectivity has not been verified in Haemaphysalis longicornis, which is a widely distributed tick species, especially in East Asian countries. Therefore, the purpose of this study was to evaluate the effectivity of subolesin vaccination against H. longicornis in a rabbit model. METHODS: Haemaphysalis longicornis (Okayama strain, female, adult, parthenogenetic strain) and Japanese white rabbits were used as the model tick and animal, respectively. The whole open reading frame of H. longicornis subolesin (HlSu) was identified and expressed as a recombinant protein using E. coli. The expression was verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the immunogenicity of rHlSu against anti-H. longicornis rabbit serum was confirmed using Western blotting. After vaccination of rHlSu in rabbits, experimental infestation of H. longicornis was performed. Variables related to blood-feeding periods, pre-oviposition periods, body weight at engorgement, egg mass, egg mass to body weight ratio, and egg-hatching periods were measured to evaluate the effectiveness of subolesin vaccination. RESULTS: The whole open reading frame of HlSu was 540 bp, and it was expressed as a recombinant protein. Vaccination with rHlSu stimulated an immune response in rabbits. In the rHlSu-vaccinated group, body weight at engorgement, egg mass, and egg mass to body weight ratio were statistically significantly lower than those in the control group. Besides, egg-hatching periods were extended significantly. Blood-feeding periods and pre-oviposition periods were not different between the two groups. In total, the calculated vaccine efficacy was 37.4%. CONCLUSIONS: Vaccination of rabbits with rHlSu significantly affected the blood-feeding and reproduction in H. longicornis. Combined with findings from previous studies, our findings suggest subolesin has the potential to be used as a universal tick vaccine.

ZnO nanorod arrays assembled on activated carbon fibers for photocatalytic degradation: Characteristics and synergistic effects.

Well-aligned ZnO nanorod arrays were assembled on activated carbon fibers by a stepwise sequence of sol-gel and hydrothermal synthesis methods. These ZnO nanorod arrays on activated carbon fibers having different characteristics such as surface area, rod concentration, aspect ratio and defect level, were applied as catalysts for the photodegradation of an aqueous methylene blue solution. They showed very promising methylene blue adsorbility in the dark (ca. 0.025-0.031 mg methylene blue m-2 catalyst, vs. 0.072 mg methylene blue m-2 activated carbon fibers). Significantly, the defect level of ZnO nanorod arrays has a major effect on the turnover frequency compared to other characteristics. A synergistic effect between activated carbon fibers and ZnO nanocrystals on enhancing turnover frequency was more significant for the well-assembled ZnO nanorod arrays on activated carbon fibers catalysts compared to the mechanically mixed ZnO powder with activated carbon fibers catalyst. Further, turnover frequency for the ZnO nanorod arrays on activated carbon fibers (0.00312 molmethylene blue molZnO-1 h-1) was twice higher than that for the corresponding bare ZnO nanorod arrays, and 3 times higher than that for a commercial ZnO powder. In addition, ZnO nanorod arrays on activated carbon fibers show high degradation (77.5%) and mineralization (55.0%) levels for methylene blue, and also good reusability (or stability) as demonstrated by a sequential 5-time recycle routine. These outstanding features indicate that activated carbon fibers supported ZnO nanorod arrays have significant potential to be used as catalysts for photodegradation.

Porin Expression Profiles in Haemaphysalis longicornis Infected With Babesia microti.

The porin gene is widely disseminated in various organisms and has a pivotal role in the regulation of pathogen infection in blood-sucking arthropods. However, to date, information on the porin gene from the Haemaphysalis longicornis tick, an important vector of human and animal diseases, remains unknown. In this study, we identified the porin gene from H. longicornis and evaluated its expression levels in Babesia microti-infected and -uninfected H. longicornis ticks at developmental stages. We also analyzed porin functions in relation to both tick blood feeding and Babesia infection and the relationship between porin and porin-related apoptosis genes such as B-cell lymphoma (Bcl), cytochrome complex (Cytc), caspase 2 (Cas2), and caspase 8 (Cas8). The coding nucleotide sequence of H. longicornis porin cDNA was found to be 849 bp in length and encoded 282 amino acids. Domain analysis showed the protein to contain six determinants of voltage gating and two polypeptide binding sites. Porin mRNA levels were not significantly different between 1-day-laid and 7-day-laid eggs. In the nymphal stage, higher porin expression levels were found in unfed, 12-h-partially-fed (12 hPF), 1-day-partially-fed (1 dPF), 2 dPF nymphs and nymphs at 0 day post-engorgement (0 dAE) vs. nymphs at 2 dAE. Cytc and Cas2 mRNA levels were higher in 2 dPF nymphs in contrast to nymphs at 2 dAE. Porin expression levels appeared to be higher in the infected vs. uninfected nymphs during blood feeding except at 1 dPF and 0-1 dAE. Especially, the highest B. microti burden negatively affected porin mRNA levels in both nymphs and female adults. Porin knockdown affected body weight and Babesia infection levels and significantly downregulated the expression levels of Cytc and Bcl in H. longicornis female ticks. In addition, this study showed that infection levels of the B. microti Gray strain in nymphal and female H. longicornis peaked at or around engorgement from blood feeding to post engorgement. Taken together, the research conducted in this study suggests that H. longicornis porin might interfere with blood feeding and B. microti infection.

Identification of Haemaphysalis longicornis Genes Differentially Expressed in Response to Babesia microti Infection.

Haemaphysalis longicornis is a tick and a vector of various pathogens, including the human pathogenetic Babesia microti. The objective of this study was to identify female H. longicornis genes differentially expressed in response to infection with B. microti Gray strain by using a suppression subtractive hybridization (SSH) procedure. A total of 302 randomly selected clones were sequenced and analyzed in the forward subtracted SSH cDNA library related to Babesia infection, and 110 clones in the reverse cDNA library. Gene ontology assignments and sequence analyses of tick sequences in the forward cDNA library showed that 14 genes were related to response to stimulus or/and immune system process, and 7 genes had the higher number of standardized sequences per kilobase (SPK). Subsequent real-time PCR detection showed that eight genes including those encoding for Obg-like ATPase 1 (ola1), Calreticulin (crt), vitellogenin 1 (Vg1) and Vg2 were up-regulated in fed ticks. Compared to uninfected ticks, infected ticks had six up-regulated genes, including ola1, crt and Vg2. Functional analysis of up-regulated genes in fed or Babesia-infected ticks by RNA interference showed that knockdown of crt and Vg2 in infected ticks and knockdown of ola1 in uninfected ticks accelerated engorgement. In contrast, Vg1 knockdown in infected ticks had delayed engorgement. Knockdown of crt and Vg1 in infected ticks decreased engorged female weight. Vg2 knockdown reduced B. microti infection levels by 51% when compared with controls. The results reported here increase our understanding of roles of H. longicornis genes in blood feeding and B. microti infection.

Transient Transfection of the Zoonotic Parasite Babesia microti.

The development of genetic manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not been established for Babesia microti. Here, we report the first successful transient transfection of B. microti. The plasmids containing the firefly luciferase reporter gene were transfected into B. microti by an AMAXA 4D Nucleofection system. Twenty-four-hour synchronization, the 5'-actin promoter, program FA100, and 50 µg of plasmid DNA constituted the best conditions for the transient transfection of B. microti. This finding is the first step towards a stable transfection method for B. microti, which may contribute to a better understanding of the biology of the parasite.