16S rRNA metabarcoding for the identification of tick-borne bacteria in ticks in the Republic of Korea.

Ticks are blood-sucking ectoparasites that act as vectors for transmission of various pathogens. The purpose of this study was to assess tick-borne bacteria, whether pathogenic or not, in ticks distributed in Korea using 16S rRNA metabarcoding and to confirm the results by PCR. Questing ticks were collected from four provinces in Korea in 2021 using the flagging method. After pooling the DNAs from the 61 tick pools (including 372 ticks), the bacterial 16S rRNA V3-V4 hypervariable region was amplified and sequenced using the MiSeq platform. Rickettsia, Ehrlichia, and the endosymbiont Wolbachia were confirmed by conventional PCR and molecular analysis. In total, 6907 ticks (534 pools) were collected and identified as belonging to five species (Haemaphysalis spp., H. longicornis, H. flava, I. nipponensis, and A. testudinarium). Through 16S rRNA metabarcoding, 240 amplicon sequence variants were identified. The dominant taxa were Rickettsiella and Coxiella. Additionally, pathogenic bacteria such as Rickettsia and Ehrlichia, endosymbiotic bacteria such as Wolbachia and Spiroplasma were identified. Polymerase chain reaction (PCR) was performed to confirm the presence of Rickettsia, Ehrlichia, Bartonella, and Wolbachia in individual ticks. Overall, 352 (65.92%) of 534 pools tested positive for at least one of the screened tick-borne bacteria. Rickettsia was the most prevalent (61.42%), followed by Wolbachia (5.05%). Ehrlichia was detected in 4.86% of tested samples, whereas Bartonella was not detected. In this study, 16S rRNA metabarcoding revealed the presence of Rickettsia, Wolbachia, and Ehrlichia, in that order of abundance, while showing absence of Bartonella. These results were confirmed to exhibit the same trend as that of the conventional PCR. Therefore, large-scale screening studies based on pooling, as applied in this study, will be useful for examining novel or rare pathogens present in various hosts and vectors.

The detection of zoonotic microorganisms in Rhipicephalus sanguineus (brown dog ticks) from Vietnam and the frequency of tick infestations in owned dogs.

Dog owners are greatly concerned about tick infestations in their pets. The prevalence and dispersion of ticks and their disease-causing microorganisms have been limited from the viewpoint of dog owners in Vietnam. This study investigated the presence of tick infestation and the pathogens associated with it in canines that were brought to veterinary hospitals in Vietnam. In the survey, 1,423 dogs participated from February to October 2022. Molecular and morphological methods were utilized to identify ticks and the associated pathogens. In addition,risk variables linked to tick infestation were documented and analyzed using statistical methods. The total exposure to the brown dog tick (Rhipicephalus sanguineus sensu lato) was 29.01%. Nam Dinh has the highest tick prevalence among the research areas. Tick infestation reached its highest point between June and September in the northern region of the country, with distinct seasons showing a strong correlation with tick infestation in dogs. Out of 177 tick pools examined, 146(82.49%) tested positive for at least one infection. Mycoplasma spp. (78.53%) was the most common, followed by Anaplasma spp. (37.29%), Rickettsia felis (5.08%), Babesia vogeli, and Hepatozoon canis (2.82%). In the current study, there was a statistically significant link between tick infestation and characteristics such as age, breed, body size, lifestyle, and bathing frequency. Understanding the seasonal behavior of vector ticks is crucial for identifying individuals or animals susceptible to tick-borne diseases. Studying the distribution of ticks and their ability to carry and disseminate zoonotic germs in specific places could assist veterinarians and policymakers in implementing effective strategies to manage zoonotic infections.

Acaricidal activity of Egyptian crude plant extracts against Haemaphysalis longicornis ticks.

Haemaphysalis longicornis is a common Ixodida tick species found in temperate areas of Asian countries. An anti-tick assay was conducted on adult female H. longicornis ticks. Plant extract solutions were prepared at concentrations of 50, 25, and 10 mg/mL. Tick survival and mortality were assessed by counting the number of dead and live ticks at 24 h, 48 h, 72 h, and 96 h posttreatment. Out of 11 plant extracts screened, Artemisia judaica extract exhibited the highest potency with 100% mortality (5/5) at 48 h when applied at high and moderate concentrations (50 and 25 mg/mL). Similar results were observed at 96 h for the 10 mg/mL group compared to the untreated ticks. Cleome droserifolia extract demonstrated partial activity with 60% (3/5) and 20% (1/5) mortality at 96 h posttreatment at concentrations of 50 and 25 mg/mL, respectively. Forsskaolea tenacissima extract showed a weak effect with 100% tick mortality (5/5) only at the highest treatment concentration after 96 h. To confirm the activity of A. judaica, trial 2 was conducted. A. judaica demonstrated potency within 48 h in high dose and 72 h in moderate dose, with 100% mortality (15/15) at 96 h posttreatment compared to untreated ticks. The median lethal time 50 (LT50) values were 30.37 h for the high and 55.08 h for the moderate doses. Liquid chromatography‒mass spectrometry was performed on the most potent candidate (A. judaica) to identify its phytochemical components. The results revealed the presence of 9 compounds identified through manual annotation and 74 compounds from the Global Natural Products Social library. These compounds included terpenoids, steroids, phenylpropanoids, flavonoid glycosides, flavonoids, and benzenoids. Camphor was identified as the major component via both approaches. These findings suggest the potential use of A. judaica extract in the future development of acaricidal therapeutics.

Acaricidal activity of Erigeron acer L. root against Haemaphysalis longicornis and phytochemical profiling by liquid chromatography-tandem mass spectrometry.

The present study is focused on evaluating acaricidal activity and chemical compositions of Erigeron acer root, which was identified as a promising candidate among fifteen Mongolian plant extracts tested for acaricidal activity. The acaricidal effect was evaluated against Haemaphysalis longicornis, assessed for toxicity to normal human skin fibroblast, and analyzed for its chemical constituents. The acetone extract of E. acer root showed significant activity against H. longicornis, with a lethal concentration (LC50) of 5.31 mg/mL and low toxicity, evidenced by a cytotoxic concentration (CC50) of 267.00 µg/mL. Using liquid chromatography-tandem mass spectrometry and molecular networking, thirteen natural compounds were identified, including pyrrolidines, alkaloids, fatty acids, and flavonoids, highlighting the efficacy of E. acer root extract as an effective acaricide against H. longicornis and offering insights for developing new tick control solutions.

First molecular survey of tick-borne protozoan and bacterial pathogens in the questing tick population in Bangladesh.

Questing ticks carry various tick-borne pathogens (TBPs) that are responsible for causing tick-borne diseases (TBDs) in humans and animals around the globe, especially in the tropics and sub-tropics. Information on the distribution of ticks and TBPs in a specific geography is crucial for the formulation of mitigation measures against TBDs. Therefore, this study aimed to survey the TBPs in the questing tick population in Bangladesh. A total of 2748 questing hard ticks were collected from the pastures in Sylhet, Bandarban, Sirajganj, Dhaka, and Mymensingh districts through the flagging method. After morphological identification, the ticks were grouped into 142 pools based on their species, sexes, life stages, and collection sites. The genomic DNA extracted from tick specimens was screened for 14 pathogens, namely Babesia bigemina (AMA-1), Babesia bovis (RAP-1), Babesia naoakii (AMA-1), Babesia ovis (18S rRNA), Theileria luwenshuni (18S rRNA), Theileria annulata (Tams-1), Theileria orientalis (MPSP), Anaplasma marginale (groEL), Anaplasma phagocytophilum (16S rRNA), Anaplasma bovis (16S rRNA), Anaplasma platys (16S rRNA), Ehrlichia spp. (16S rRNA), Rickettsia spp. (gltA), and Borrelia (Bo.) spp. (flagellin B) using genus and species-specific polymerase chain reaction (PCR) assays. The prevalence of the detected pathogens was calculated using the maximum likelihood method (MLE) with 95 % confidence interval (CI). Among 2748 ixodid ticks, 2332 (84.86 %) and 416 (15.14 %) were identified as Haemaphysalis bispinosa and Rhipicephalus microplus, respectively. Haemaphysalis bispinosa was found to carry all the seven detected pathogens, while larvae of R. microplus were found to carry only Bo. theileri. Among the TBPs, the highest detection rate was observed in A. bovis (20/142 pools, 0.81 %, CI: 0.51-1.20), followed by T. orientalis (19/142 pools, 0.72 %, CI: 0.44-1.09), T. luwenshuni (9/142 pools, 0.34 %, CI: 0.16-0.62), B. ovis (4/142 pools, 0.15 %, CI: 0.05 - 0.34) and Bo. theileri (4/142 pools, 0.15 %, CI: 0.05-0.34), Ehrlichia ewingii (3/142 pools, 0.11 %, CI: 0.03-0.29), and Babesia bigemina (1/142, 0.04 %, CI: 0.00 - 0.16). This study reports the existence of T. luwenshuni, E. ewingii, and Bo. theileri in Bangladesh for the first time. The novel findings of this study are the foremost documentation of transovarian transmission of B. bigemina and E. ewingii in H. bispinosa and also provide primary molecular evidence on the presence of E. ewingii and Bo. theileri in H. bispinosa. Therefore, this study may shed light on the circulating TBPs in ticks in the natural environment and thereby advocate awareness among physicians and veterinarians to control and prevent TBDs in Bangladesh.

First report of dog ticks and tick-borne pathogens they are carrying in Malawi.

Ticks are vectors for transmitting tick-borne pathogens (TBPs) in animals and humans. Therefore, tick identification is necessary to understand the distribution of tick species and the pathogens they carry. Unfortunately, data on dog ticks and the TBPs they harbor in Malawi are incomplete. This study aimed to identify dog ticks and the TBPs they transmit in Malawi. One hundred thirty-two ticks were collected from 87 apparently healthy but infested domestic dogs in four districts of Malawi, which were pooled into 128 tick samples. The ticks were morphologically identified under a stereomicroscope using identification keys, and species identification was authenticated by polymerase chain reaction (PCR) through the amplification and sequencing of 12S rRNA and cytochrome c oxidase subunit I (CO1) genes. The tick species identified were Rhipicephalus sanguineus sensu lato (58.3%), Haemaphysalis elliptica (32.6%), and Hyalomma truncatum (9.1%). Screening for TBPs using species-specific PCR assays revealed that 48.4% of the ticks were infected with at least one TBP. The TBP detection rates were 13.3% for Anaplasma platys, 10.2% for Babesia rossi, 8.6% for B. vogeli, 6.3% for Ehrlichia canis, 3.9% for A. phagocytophilum, 3.1% for B. gibsoni, 2.3% for B. canis and 0.8% for Hepatozoon canis. Co-infections of up to three pathogens were observed in 48.4% of the positive samples. This is the first study to identify dog ticks and the TBPs they harbor in Malawi. These findings provide the basis for understanding dog tick distribution and pathogens they carry in Malawi. This study necessitates the examination of ticks from more study locations to have a better picture of tick challenge, and the development of ticks and tick-borne disease control methods in Malawi.

Molecular survey of canine tick-borne pathogens in ticks and stray dogs in Dhaka city, Bangladesh.

Molecular surveillance of canine tick-borne pathogens (TBPs) in Bangladesh has constantly been undervalued. Therefore, the emergence of new pathogens often remains undetected. This study aimed to screen tick-borne pathogens in stray dogs and ticks in the Dhaka metropolitan area (DMA). Eighty-five dog blood and 53 ticks were collected in six city districts of DMA from September 2022 to January 2023. The ticks were identified by morphology. Screening of TBPs was performed by polymerase chain reaction (PCR), followed by sequencing. The PCR assays were conducted to analyze the 18S rRNA (Babesia gibsoni, B. vogeli, and Hepatozoon canis), 16S rRNA (Anaplasma phagocytophilum, A. platys, and A. bovis), gltA (Ehrlichia canis and Rickettsia spp.), flagellin B (Borrelia spp.) and 16-23S rRNA (Bartonella spp.). Three tick species, Rhipicephalus sanguineus (50/53), R. microplus (1/53), and Haemaphysalis bispinosa (2/53), were identified. Babesia gibsoni (38 out of 85) and A. platys (7 out of 85) were detected in dog blood. In contrast, four pathogens, B. gibsoni (1 out of 53), B. vogeli (1 out of 53), H. canis (22 out of 53), and A. platys (1 out of 53), were detected in the ticks. However, the detection rates of TBPs in dog blood and ticks were not correlated in this study. The phylogenetic analyses suggested that a single genotype for each of the four pathogens is circulating in DMA. This study reports the existence of B. vogeli, H. canis, and A. platys in Bangladesh for the first time.

Molecular Identification of Piroplasmids in Ticks from Infested Small Ruminants in Konya Province, Turkey.

Ticks play a pivotal role in propagating a diverse spectrum of infectious agents that detrimentally affect the health of both humans and animals. In the present study, a molecular survey was executed of piroplasmids in ticks collected from small ruminants in four districts within Konya province, Turkey. Microscopic examination identified 1281 adult ticks, which were categorized into 357 pools based on their species, sexes, host animals, and collection site before DNA extraction. The infection rates were calculated by using a maximum likelihood estimate (MLE) with 95% confidence intervals (CI). Hyalomma detritum, H. excavatum, Rhipicephalus bursa, R. sanguineus, and R. turanicus were identified in this study. Among the five tick species identified here, R. turanicus exhibited the highest infestation rate in both goats and sheep. The presence of Babesia ovis and Theileria ovis based on 18S rRNA was confirmed using molecular assay. The overall MLE of infection rates for B. ovis and T. ovis was 2.49% (CI 1.72-3.46) and 1.46% (CI 0.87-2.23), respectively. The MLE of B. ovis and T. ovis infection rates in R. bursa was 10.80% (CI 7.43-14.90) and 0.33% (CI 0.02-1.42), respectively, while that in R. turanicus was 0.12% (CI 0.01-0.51) and 2.08% (CI 1.25-3.22). This study further confirms that R. turanicus and R. sanguineus can act as vectors for B. ovis, thus advancing our comprehension of tick-borne piroplasmids epidemiology and providing valuable insights for the development of effective control strategies for ticks and tick-borne diseases in Turkey.

Draft genome sequence data of Haemaphysalis longicornis Oita strain.

Haemaphysalis longicornis Neumann, 1901 is one of the most well-known hard ticks because of its medical and veterinary importance. Haemaphysalis longicornis transmit a wide range of pathogens among vertebrates, affecting humans and animals in Asia and Oceania. In Japan, the tick species is a major pest of cattle because it can spread a protozoan parasite Theileria orientalis, which causes theileriosis and produces economic losses to the livestock industry (Yokoyama et al. 2012 [1]). Apart from bovine theileriosis, H. longicornis is a vector of bovine babesiosis caused by Babesia ovata, canine babesiosis caused by Babesia gibsoni, and rickettsiosis and viral diseases in humans. Its habitats are mainly Japan, Australia, New Zealand, New Caledonia, the Fiji Islands, Korea, China, and Russia (Oliver et al. 1973 [2]). In the United States, heavy H. longicornis infestations on cattle and white-tailed deer were reported in 2019, making it now one of the tick species to be an increasing threat to livestock animals and humans globally. Ticks reproduce offspring after mating with female and male ticks, however, interestingly, there are two races of H. longicornis: bisexual (diploid) and parthenogenetic (triploid) races [2]. Parthenogenetic H. longicornis is distributed throughout Japan, while the northern limit of the bisexual race is believed to be Fukushima Prefecture on Honshu Island (Fujita et al. 2013 and Kitaoka et al. 1961 [3,4]). This tick species is also considered to be of great scientific importance, and the parthenogenetic race collected in Okayama prefecture has been reared since 1961, while the bisexual race collected in Oita prefecture has been reared since 2008 under laboratory conditions in Japan (Boldbaatar et al. 2010 and Fujisaki et al. 1976 [5,6]). Namely, the "Okayama strain" and "Oita strain" of H. longicornis have been maintained for more than six decades and 15 years, respectively, stably under laboratory conditions. To obtain reference data of bisexual H. longicornis, we sequenced unfed females with haploid genomes using Illumina and MinION Q20 kit then obtained a draft genome consisting of 2.48 Gbp. The number of the contig was 98,529 and N50 was 46.5 Kb. Genome information derived from our laboratory colony of bisexual H. longicornis ticks would provide fundamental insight into understanding how different reproductive lineages occur within the same species of the tick.

Investigation of vertical and horizontal transmission of Spiroplasma in ticks under laboratory conditions.

Many arthropods harbour bacterial symbionts, which are maintained by vertical and/or horizontal transmission. Spiroplasma is one of the most well-known symbionts of ticks and other arthropods. It is still unclear how Spiroplasma infections have spread in tick populations despite its high prevalence in some tick species. In this study, Ixodes ovatus, which has been reported to harbour Spiroplasma ixodetis at high frequencies, was examined for its vertical transmission potential under experimental conditions. Next, two isolates of tick-derived Spiroplasma, S. ixodetis and Spiroplasma mirum, were experimentally inoculated into Spiroplasma-free Haemaphysalis longicornis colonies and the presence of Spiroplasma in their eggs and larvae was tested. Our experimental data confirmed that S. ixodetis was transmitted to eggs and larvae in a vertical manner in the original host I. ovatus. In the second experiment, there was no significant difference in engorged weight, egg weight, and hatching rate between Spiroplasma-inoculated and control H. longicornis groups. This suggested that Spiroplasma infection does not affect tick reproduction. Spiroplasma DNA was only detected in the eggs and larvae derived from some individuals of S. ixodetis-inoculated groups. This has demonstrated the potential of horizontal transmission between different tick species. These findings may help understand the transmission dynamics of Spiroplasma in nature and its adaptation mechanism to host arthropod species.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro.

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

RNA activation in ticks.

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3'- untranslated region (3'-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3'-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.

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.

Localization of secreted ferritin (FER2) in the embryos of the tick Haemaphysalis longicornis.

Despite the absence of a blood meal, embryogenesis involves many processes that require nutrients and other essential elements, including iron. Due to the lack of an external source of these nutrients, these requirements are acquired maternally. Because of the toxic nature of iron, they are transferred through iron transport molecules such as secreted ferritin (FER2). Here we tried to follow the trail of the FER2 through indirect immunofluorescence, and we observed an apparent shift of FER2 from the germ layer at the early part of development to the appendages during the late stage of embryogenesis. FER2 is also found in the middle part of the legs of the embryo. The apparent movement not only sheds light on iron processing events during embryogenesis but also indirectly guides organogenesis in the tick.

Molecular Characterization of Octopamine/Tyramine Receptor Gene of Amitraz-Resistant Rhipicephalus (Boophilus) decoloratus Ticks from Uganda.

We previously reported the emergence of amitraz-resistant Rhipicephalus (Boophilus) decoloratus ticks in the western region of Uganda. This study characterized the octopamine/tyramine receptor gene (OCT/Tyr) of amitraz-resistant and -susceptible R. (B.) decoloratus ticks from four regions of Uganda. The OCT/Tyr gene was amplified from genomic DNA of 17 R. (B.) decoloratus larval populations of known susceptibility to amitraz. The amplicons were purified, cloned and sequenced to determine mutations in the partial coding region of the OCT/Tyr gene. The amplified R. (B.) decoloratus OCT/Tyr gene was 91-100% identical to the R. (B.) microplus OCT/Tyr gene. Up to 24 single nucleotide polymorphisms (SNPs) were found in the OCT/Tyr gene from ticks obtained from high acaricide pressure areas, compared to 8 from the low acaricide pressure areas. A total of eight amino acid mutations were recorded in the partial OCT/Tyr gene from ticks from the western region, and four of them were associated with amitraz-resistant tick populations. The amino acid mutations M1G, L16F, D41G and V72A were associated with phenotypic resistance to amitraz with no specific pattern. Phylogenetic analysis revealed that the OCT/Tyr gene sequence from this study clustered into two distinct groups that separated the genotype from high acaricide pressure areas from the susceptible populations. In conclusion, this study is the first to characterize the R. (B.) decoloratus OCT/Tyr receptor gene and reports four novel amino acid mutations associated with phenotypic amitraz resistance in Uganda. However, lack of mutations in the ORF of the OCT/Tyr gene fragment for some of the amitraz-resistant R. (B.) decoloratus ticks could suggest that other mechanisms of resistance may be responsible for amitraz resistance, hence the need for further investigation.

Effect of α-Tocopheryloxy Acetic Acid on the Infection of Mice with Plasmodium berghei ANKA In Vivo and Humans with P. falciparum In Vitro.

PURPOSE: Malarial parasites are susceptible to oxidative stress. The effects of α-tocopheryloxy acetic acid (α-TEA), a vitamin E analog, on infection by Plasmodium berghei ANKA and P. falciparum in mice and human red blood cells (RBCs), respectively, were examined in this study. METHODS: For in vivo studies in mice, RBCs infected with P. berghei ANKA were inoculated via intraperitoneal injection and α-TEA was administered to C57BL/6 J male mice after infection. The blood-brain barrier (BBB) permeability was examined by Evans blue staining in experimental cerebral malaria at 7 days after infection. The in vitro inhibitory effect of α-TEA on P. falciparum 3D7 (chloroquine-sensitive strain) and K1 (multidrug-resistant strain) was tested using a SYBR Green I-based assay. RESULTS: When 1.5% α-TEA was administered for 14 days after infection, 88% of P. berghei ANKA-infected mice survived during the experimental period. Nevertheless, all the control mice died within 12 days of infection. Furthermore, the Evans blue intensity in α-TEA-treated mice brains was less than that in untreated mice, indicating that α-TEA might inhibit the destruction of the BBB and progression of cerebral malaria. The in vitro experiment revealed that α-TEA inhibited the proliferation of both the 3D7 and K1 strains. CONCLUSION: This study showed that α-TEA is effective against murine and human malaria in vivo and in vitro, respectively. Although α-TEA alone has a sufficient antimalarial effect, future research could focus on the structure-activity relationship to achieve better pharmacokinetics and decrease the cytotoxicity and/or the combined effect of α-TEA with existing drugs. In addition, the prophylactic antimalarial activity of premedication with α-TEA may also be an interesting perspective in the future.

Vitellogenin-2 Accumulation in the Fat Body and Hemolymph of Babesia-Infected Haemaphysalis longicornis Ticks.

The protozoan parasite Babesia spp. invades into tick oocytes and remains in the offspring. The transovarial transmission phenomenon of Babesia in ticks has been demonstrated experimentally, but the molecular mechanisms remain unclear. Babesia invasion into oocytes occurs along with the progression of oogenesis. In the present study, to find the key tick factor(s) for Babesia transmission, we focused on molecules involved in yolk protein precursor (vitellogenin, Vg) synthesis and Vg uptake, which are crucial events in tick oogenesis. With a Haemaphysalis longicornis tick-Babesia ovata experimental model, the expression profiles of Akt, target of rapamycin, S6K, GATA, and Vg, Vg synthesis-related genes, and Vg receptor (VgR) and autophagy-related gene 6 (ATG6), Vg uptake-related genes, were analyzed using real-time PCR using tissues collected during the preovipositional period in Babesia-infected ticks. The expression levels of H. longicornis Vg-2 (HlVg-2) and HlVg-3 decreased in the fat body of Babesia-infected ticks 1 day after engorgement. In the ovary, HlVg-2 mRNA expression was significantly higher in Babesia-infected ticks than in uninfected ticks 1 and 2 days after engorgement and decreased 3 days after engorgement. HlVgR expression was significantly lower in Babesia-infected ticks than in uninfected ticks 2 and 4 days after engorgement. HlATG6 had a lower gene expression in Babesia-infected ticks compared to uninfected ticks 2 days after engorgement. Additionally, western blot analysis using protein extracts from each collected tissue revealed that H. longicornis Vg-2 (HlVg-2) accumulate in the fat body and hemolymph of Babesia-infected ticks. These results suggest that Vg uptake from the hemolymph to the ovary was suppressed in the presence of B. ovata. Moreover, HlVg-2 knockdown ticks had a lower detection rate of B. ovata DNA in the ovary and a significant reduction of B. ovata DNA in the hemolymph compared with control ticks. Taken together, our results suggest that accumulated HlVg-2 is associated with Babesia infection or transmission in the tick body. These findings, besides previous reports on VgR, provide important information to elucidate the transovarial transmission mechanisms of pathogens in tick vectors.

Protozoan and Rickettsial Pathogens in Ticks Collected from Infested Cattle from Turkey.

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05−100% nucleotide identity with those deposited in GenBank (query cover range: 89−100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey.

Molecular Identification of Selected Tick-Borne Protozoan and Bacterial Pathogens in Thoroughbred Racehorses in Cavite, Philippines.

Tick-borne diseases (TBDs) considerably impair equine health and productivity. Moreover, TBDs, particularly equine piroplasmosis, impede international movement and trade of equids, which is a vital component of the global horse racing industry. In the Philippines, horse racing is a lucrative industry generating millions of USD annually. However, information on equine TBDs is scarce. This study intended to describe molecularly the equine tick-borne infections in a racehorse park in Cavite, Philippines and identify the risk factors associated with the infections. One hundred twenty-four (n = 124) thoroughbred racehorses were sampled and screened for selected tick-borne protozoan and bacterial pathogens using polymerase chain reaction (PCR) assays. Racehorses were positive for Babesia caballi (12.10%; 15/124), Theileria equi (0.81%; 1/124), Anaplasma phagocytophilum (10.48%; 13/124), Borrelia burgdorferi sensu lato (38.71%; 48/124), A. marginale (0.81%; 1/124), and Coxiella burnetii (0.81%; 1/124). Rickettsia was not detected in the samples. Gender was determined as a significant risk factor for B. caballi infection. Sequencing analysis revealed that seven partial 18S rRNA B. caballi isolates shared 98.63-100% identity with each other and were classified as genotype A. Meanwhile, the sequence obtained from the lone T. equi-positive sample was 99.77% identical to isolates from Spain, Switzerland, China, Saudi Arabia, and South Korea, and was confirmed as genotype E based on the 18S rRNA gene. Eight Anaplasma 16S rRNA partial sequences were highly identical to A. phagocytophilum and A. ovis. Partial sequences of Borrelia 5-23S rRNA were most closely related to B. japonica and other Borrelia sp. isolates from various countries. This study reports the first molecular detection of Borrelia and Anaplasma and the identification of B. caballi and T. equi genotypes in racehorses in the Philippines. Findings from this study shall be useful in crafting equine tick and TBD control and prevention programs in the country.

Data from expressed sequence tags from the organs and embryos of parthenogenetic Haemaphysalis longicornis.

OBJECTIVES: Haemaphysalis longicornis is the most important tick species in Japan and has a wide range of vector capacity. Due to its veterinary and medical importance, this tick species has been used as a model for tick/vector biological studies. To identify the key molecules associated with physiological processes during blood feeding and embryogenesis, full-length cDNA libraries were constructed using the fat body, hemocytes-containing hemolymph, midgut, ovary and salivary glands of fed females and embryos of the laboratory colony of parthenogenetic H. longicornis. The sequences of cDNA from the salivary glands had been already released. However, the related information is still poor, and the other expressed sequence tags have not yet been deposited. DATA DESCRIPTION: A total of 39,113 expressed sequence tags were obtained and deposited at the DNA DataBank of Japan. There were 7745 sequences from embryos, 7385 from the fat body, 8303 from the hemolymph including hemocytes, 7385 from the midgut, and 8295 from the ovary. The data, including expressed sequence tags from the salivary glands was summarized into Microsoft Excel files. Sharing this data resource with the tick research community will be valuable for the identification of novel genes and advance the progress of tick research.