Aphidius ervi, display significant differences in terms of host preference and host acceptance, depending on the host on which they developed , which is preferred over a non-natal host, a trait known as host fidelity. This trait allows females to quickly find hosts in heterogeneous environments, a process mediated by chemosensory/olfactory mechanisms, as parasitoids rely on olfaction and chemical cues during host selection. Thus, it is expected that proteins participating in chemosensory recognition, such as odorant-binding proteins (OBPs) and odorant receptors (ORs) would play a key role in host preference. In this study, we addressed the effect of parasitoid reciprocal host switching between two aphid hosts (Sitobion avenae and Acyrthosiphon pisum) on the expression patterns of chemosensory genes in the wasp A. ervi. First, by using a transcriptomic approach based on RNAseq of A. ervi females reared on S. avenae and A. pisum, we were able to annotate a total of 91 transcripts related to chemoperception. We also performed an in-silico expression analysis and found three OBPs and five ORs displaying different expression levels. Then, by using qRT-PCR amplification, we found significant differences in the expression levels of these eight genes when the parasitoids were reciprocally transplanted from S. avenae onto A. pisum and vice versa. This suggests that the expression levels of genes coding for odorant receptors and odorant-binding proteins would be regulated by the specific plant–aphid host complex where the parasitoids develop  and that chemosensory genes coding for olfactory mechanisms would play a crucial role on host preference and host acceptance, ultimately leading to the establishment of host fidelity in A. ervi parasitoids.Generalist parasitoids of aphids, such as the wasp  Aphidius ervi Haliday (Hymenoptera: Braconidae), a worldwide distributed koinobiont parasitoid of several aphid species [A. ervi mainly parasitizes the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) [Sitobion avenae Fabricius (Hemiptera: Aphididae) [A. ervi in biological control programs depends on the specificity of host selection and host acceptance behaviors and on host suitability, as not all host species are suitable for parasitoids [A. ervi is intimately related to behavioral and foraging strategies used during the selection and parasitization of a suitable host [Parasitoid wasps are a diverse group of hymenopterans that are natural enemies of a broad range of arthropods, including those of agronomic significance . Adult p species . Extensihididae) ,6, althohididae) ,8. Howevasitoids . Thus, tble host .Interestingly, maternal previous experience plays a key role in determining the host choice in the offspring, causing transgenerational phenotypic plasticity or maternal effect . The oviHost finding relies on the olfaction of environmental signals, such as specific chemicals and volatiles, emitted either from plants or from a host–plant complex, which are used by female wasps to carefully choose an appropriate host for oviposition ,17,18. HIn adult parasitoids, it has been proposed that host preference would be a consequence of exposure, during larval stages, to both host- and host–plant-related chemical volatiles and cues  which are emitted by the plant–host complex upon aphid infestation  and whicGiven that olfactory behavioral responses depend on specific sets of proteins for odorant recognition and signal propagation and processing, it has been proposed that there is a molecular base underpinning the phenotypic plasticity of behavioral responses displayed by insects towards olfactory signals and cues . Thus, vA. ervi differ in several biological aspects  [A. ervi lineages reared on natal and non-natal hosts. If A. ervi uses the same strategy  for parasitizing both hosts, then the expression of chemosensory genes should be very similar. Alternatively, if A. ervi is able to modulate its olfactory system to encompass the composition of novel odorants and kairomones, then differences in terms of expression of chemosensory genes should be detected.However, as the aphid species parasitized by s, etc.) , it is nA. ervi populations that naturally parasitize different aphid species (A. pisum and S. avenae). First, we used a transcriptomic approach based on RNAseq, to identify putative transcripts related to chemoperception in A. ervi. This approach allowed us to annotate chemosensory genes and characterize their expression levels when the parasitoids were reared on two different aphid hosts. Then, we studied if the exposure to a non-natal plant–host complex (regardless of the parasitoid lineage) had an impact on the expression of genes coding for odorant receptors (ORs) and odorant-binding proteins . Thus, we compared the effects of host change from the natal aphid host to an alternative non-natal host on specific chemosensory genes, in order to outline the molecular mechanism underlying host fidelity establishment. Finally, as parasitoid wasps are usually reared in caged conditions before being released for biological control programs, which has been shown to increase inbreeding and reduce host fidelity [A. ervi parasitoids sampled from field (exogamic) and laboratory (endogamic) populations.In this study, we addressed the effect of reciprocal host switching between aphid host species on the expression patterns of chemosensory genes on two fidelity , we compA. pisum and S. avenae were collected as aphid mummies from fields of alfalfa (Medicago sativa L.) and wheat (Triticum aestivum L.), respectively, in Region del Maule, Chile . Aphid mummies were individually isolated in Petri dishes until adult parasitoid emergence. The emerged naïve parasitoids were then identified as A. ervi and sexed following standard taxonomic keys [A. ervi parasitoids were founded from five naïve A. ervi virgin females and one naïve virgin male selected at random and obtained from the same aphid host population from which they were collected (A. pisum or S. avenae). Female and male individuals were left to mate in a Petri dish for 24 h with diluted honey and water for sustenance. Mated females were then transferred to a cage containing aphids ad libitum from the same species from which they emerged, with diluted honey and water for sustenance. The establishment of A. ervi parasitoids on their natal host for one single generation has been shown to erase any previous field experience : (i) one A. ervi (Ae) population from A. pisum  maintained on broad bean (Vicia faba L.)  and (ii) one A. ervi population from S. avenae (SA) maintained on barley (Hordeum vulgare L.) . These aphids and their host plants have been used successfully for A. ervi rearing in previous studies [Hamiltonella defensa, which confers protection against parasitoids [Parasitized individuals of mic keys  under annce (see ). Thus,  studies ,31,33. A studies ,35, inclasitoids ,37.A. ervi reference transcriptome (available at 10.6084/m9.figshare.4816939). Briefly, RNA was extracted from dissected heads and bodies of 60 female A. ervi parasitoids, which were collected alive from three caged parasitoid populations . For further details of the experiment, see reference [A. ervi sequenced samples. The remaining RNA was used for library construction using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina) and sequenced using an Illumina HiSeq 2000 . The raw RNA-seq libraries used in this study are available in NCBI . Raw reads were assembled into a reference A. ervi transcriptome using Trinity 2.0.6 and annotated with BLASTx using the NR database (April 2016). Further details on both assembly and annotation are published elsewhere [In this study, we used the eference . After clsewhere .A. ervi transcriptomic annotation table , which have been functionally characterized and for which odor response data are available , we per2069.v1; . Briefly2069.v1; ) and cou2069.v1; . Then, a2069.v1; . Hence, /tissues . All che. pisum; ) were covailable .A. ervi, a reciprocal transplant experiment was conducted  [A. ervi wasps [Aphid mummies from the third generation of each condition  were isolated in Petri dishes until parasitoid emergence. Since mated females display a higher attraction to oviposition-site cues, virgin adult female parasitoids were left to mate with a male from the same condition for 24 h . Each ma avenae) . After svi wasps ,31.A. ervi parasitoids acclimated on natal or non-natal hosts for two generations (exogamic population) with A. ervi parasitoids from inbred populations that had been maintained in the laboratory on the same natal plant–host complex for more than 75 generations (endogamic population)  on the expression of chemosensory genes, we compared field populations of ulation) . The expWe decided to study heads, as they contain most of the organs involved in chemosensory function and feeding, as well as most of the olfactory-associated proteins ,48. FemaA. ervi females transplanted to their natal or non-natal hosts (A. ervi transcriptome to retrieve template sequences [A. ervi genomic DNA). All PCR reactions were carried out in triplicate  using the Mx3000 P qPCR system  under the following cycling conditions: 95 °C for 10 min, 40 cycles of 95 °C for 30 s, 56 °C for 45 s, and 72 °C for 40 s. A dissociation curve was included immediately after each qPCR, using a ramp of 55–95 °C to confirm the absence of non-specific amplifications. All amplicons were sequenced to confirm the specific amplification of the target genes.Determinations of the relative transcript abundance of eight chemosensation-related genes . The expression of a given gene was compared between parasitoids reared on natal and non-natal hosts, considering the natal condition as the control. In the case of gene expression comparison between outbred and inbred A. ervi populations, the outbred condition was considered as the control.Expression data for each target gene were normalized using published primers which amplify Ribosomal Protein L19 of A. ervi  , and ORs found within the criptome . We annocriptome . Furtherid hosts .A. pisum and S. avenae that were reared on their natal host or transplanted to non-natal hosts  to SA , although no differences were observed for the reciprocal switch (SA to AP). Finally, OBP-C was downregulated when A. ervi was switched from SA to AP but not when the parasitoids were transplanted from AP to SA.The expression of target chemosensation-related genes was assessed by qRT-PCR when parasitoid females from the same natal host were reared on their natal (control condition) and non-natal  hosts. We observed a variation in the expression levels when comparing chemosensory genes of parasitoids originated from al hosts . Our resed to SA , regardlThe expression of target chemosensation-related genes was assessed by qRT-PCR when parasitoid females from different natal hosts (AP and SA) were reared on non-natal aphid host species . These cOR-E) displayed similar expression levels in parasitoids reared on SA  compared to parasitoids transplanted to SA , while three ORs were downregulated . Hence, switching parasitoids from SA to AP had a greater effect on the expression of OR genes, while the expression of OBP genes remained similar in the two conditions  and inbred laboratory caged (endogamic) populations of the parasitoid wasp for OBPs . In the pulation .Comparisons between outbred  and inbred  parasitoid populations switched to non-natal host SA displayed lower expression levels for both ORs and OBPs .A. ervi has been successfully used for biocontrol of economically relevant aphid species [A. pisum and S. avenae) [A. ervi [ species , as it h avenae) . In this avenae) ,52. Henc avenae) . Interes avenae) ,53 and m[A. ervi ,30. Alth[A. ervi ,55, it iA. ervi and identify genes displaying differential expression levels between parasitoids reared on different hosts. By conducting reciprocal transplant experiments and using quantitative PCR, we found that switching A. ervi females to a novel plant–host complex  had significant effects on the transcript abundances of chemosensation-related genes in the offspring of those females, regardless of the natal host (A. pisum and S. avenae). Surprisingly, we also observed differences in the expression profiles of ORs and OBPs when comparing field and caged parasitoids reared on the same aphid host species. The significantly lower abundance of transcripts measured for these genes might be caused by long-time inbreeding under laboratory conditions . Thus, it is possible that A. ervi inbreeding might disrupt the balance of the highly sensitive and coordinated mechanisms of olfaction, which may explain the loss of host fidelity observed previously [In this study, by using RNAseq transcriptomic information, we were able to annotate several chemosensory genes in eviously .A. ervi transcriptome assembly as a reference [A. ervi reared on S. avenae and reared on A. pisum [Olfaction and chemosensory perception are key functions for host finding and host recognition. Thus, based on the eference , we ideneference , we alsoA. pisum . A possiA. pisum ,57. TherA. pisum  would leA. pisum ,55 and mA. pisum .D. melanogaster [In insects, olfaction is triggered when odorants and other semiochemicals reach the sensillar lymph through pore tubules located in the antenna and bind to OBPs. Then, the odorant–OBP complex is transported through the sensillum lymph to their receptors on olfactory neurons . OBPs arnogaster , and maynogaster . This hanogaster ,41.Microplitis mediator [M. mediator showed that two of the OBPs analyzed in our study (OBP-A and OBP-F) had high identity values (>40%) with OBP8 and OBP10 from M. mediator, while the top blast hit for OBP-C was pheromone-binding protein 1 from M. mediator. Functional analysis of OBP8 and OBP10 in M. mediator has shown that these genes are expressed mainly in the antennae of adult wasps and can bind a broad range of odorant molecules, including nonane, farnesol, nerolidol, nonanal, β-ionone, acetic ether, and farnesene, with different binding affinities [S. avenae compared to parasitoids maintained on A. pisum  may be related to the developmental exposure of A. ervi larva to the plant–aphid host complex, as aphid mummies were taken straight from their rearing cages and isolated in Petri dishes. Moreover, exposure to host plant volatiles from infested plants during larval stages of A. ervi would induce olfactory responses in the adults [The odorant-binding properties of OBPs have been determined for different insect species , includimediator . Interesfinities . Additiofinities . The hige adults ,62.A. ervi [A. ervi due to its lack of specificity, as EBF has been reported in both S. avenae and A. pisum among other aphid species [n-alkanes are the predominant component of CHCs and may include alkenes and their methyl branches derivatives [S. avenae, n-alkanes range from C23 to C33, with three predominant compounds being n-Heptacosane , n-Nonacosane , and n-Hentriacontane  [n-alkanes are present in different proportions in A. pisum  [Parasitoid females are attracted to volatiles emitted by aphids and may use them as a host-species recognition mechanism . For insA. ervi . However species . Convers species , and are species . These v species ,68,69. H species ,71. In tivatives  but diff31, 10%) . Contras31, 21%) .Harpegnathos saltator (Hymenoptera: Formicidae), where several ORs are narrowly tuned to specific CHCs [A. ervi parasitoids reared on their natal host are not expected to respond to volatiles which they have not been previously exposed to or experienced . This is because OR-coding genes might change their regulation and expression as a response to long exposures to specific environments, for example, during the developmental time into the body of the aphid host, which should modify the offspring oviposition behavior [As host recognition in parasitoid wasps is achieved after antennal contact with kairomones and chemical cues located on the cuticle of insects , it is efic CHCs . As currfic CHCs , A. ervibehavior .OR-B, OR-E, OR-H, and OR-J) in parasitoids transplanted onto S. avenae compared with parasitoids reared on the natal host A. pisum. Furthermore, homology searches using these four ORs as queries in BLASTx alignments against Drosophila spp. found their corresponding homolog sequences -3-hexen-1-ol, which is one of the most abundant compounds found in the volatile blends emitted by V. faba plants when infested with A. pisum, and 1-Octen-3-ol, a volatile emitted by V. faba plants in response to herbivore walking activity [OR85d shows response to 6-methyl-5-hepten-2-one [A. ervi females and is found in the headspace of V. faba plants infested with A. pisum. However, the release of this compound is not induced by other aphids like the black bean aphid Aphis fabae (Hemiptera: Aphididae), a non-suitable aphid host for A. ervi [A. ervi reared on A. pisum are able to display plasticity for the expression of ORs when transplanted to a new aphid–plant complex . This downregulation observed in A. ervi transplanted from A. pisum to S. avenae could be a consequence of a reduced exposure to volatiles from the interaction between A. pisum and broad bean and suggests that the expression of chemosensory genes is indeed affected by the exposure to plant volatiles, as reported for other insect species [A. ervi transplanted from S. avenae onto A. pisum, only OR-H showed a significant upregulation, while the other ORs showed no variation. This imply that OR-H display changes in gene expression in response to new chemical cues, thus suggesting that A. ervi originated from a cereal aphid display a narrow plasticity in terms of ORs gene expression compared to parasitoids originated from a legume aphid.In our study, a significant downregulation was observed for four out of five odorant receptors  or are transplanted onto a novel aphid–plant host complex (S. avenae).Chemosensory mechanisms play a key role in insect host location and host discrimination . Howeveriginated . Hence, A. ervi were collected from alfalfa crops and maintained in an A. pisum–Vicia faba system for two generations, caged populations also sampled from alfalfa were maintained for over 2 years (>75 generations) in that same aphid–plant system [V. faba for several generations might explain the higher OR-E expression and the reduced expression of other chemosensory genes observed when field and caged populations were compared.While field populations of t system . As the t system . FurtherA. ervi populations would explain their changes in host preference behavior [The reduced expression of ORs and OBPs observed in inbred behavior , where abehavior ,31,83. Ibehavior . It is abehavior ,86. HoweOR genes are silenced, in order to verify whether host selection in A. ervi is actually based on changes in gene expression rather than on genetic differences.The loss of sensitivity and discrimination ability of the olfaction system in a laboratory-reared parasitoid wasp can have undesirable effects on the efficiency of biological control, preventing the identification of a specific target pest in agroecosystems . Hence,


Metabolomics is a powerful tool for measuring the functional output of the microbiota. Currently, there are few established workflows for analysis downstream of metabolite identification. Metabolomics is a powerful tool for measuring the functional output of the microbiota. Currently, there are few established workflows for analysis downstream of metabolite identification. Here, we introduce omu, an R package designed for assigning compound hierarchies and linking compounds to corresponding enzyme and gene annotations for organisms of interest. The omu R package is designed to analyze processed metabolomics count data. The central idea behind omu is assigning hierarchical metadata from the Kyoto Encyclopedia of Genes and Genomes (KEGG)  to each t test on each metabolite between two experimental groups, provide a fold-change value for each compound, and use these data in conjunction with the hierarchical compound annotation to create intuitive figures. The count_fold_changes function can be used to provide a count table of every metabolite within a KEGG compound category that significantly increased or decreased between groups to make bar plots  and was built using R (https://cran.r-project.org/web/packages/omu/vignettes/Omu_vignette.html.omu is available for download on the CRAN repository ( using R , devtool using R , dplyr ( using R , ggforti using R , ggplot2 using R , KEGGRES using R , knitr ( using R , magritt using R , plyr (1 using R , reshape using R , rmarkdo using R , stringr using R , roxygen using R , and tid using R . Detaile


Bou Ghanem works in the field of innate immune senescence, inflammation, and host defense. In this mSphere of Influence article, she reflects on how “Adenosine A 2B receptor deficiency promotes host defenses against Gram-negative bacterial pneumonia” by Barletta et al.  impacted her own work examining the role of the extracellular adenosine pathway in neutrophil responses and host defense against pneumococcal pneumonia.Elsa N. Bou Ghanem works in the field of innate immune senescence, inflammation, and host defense. In this mSphere of Influence article, she reflects on how “Adenosine A These findings were in contrast to acute lung injury models where A2B protected the host by modulating neutrophil recruitment   , which rococcus) . I was fococcus) , 14 and ococcus) . Importaococcus) . Surprisne cells . On the nfection . Howevernfection . As adenK. pneumoniae paper came out and following that, several papers exploring the role of the extracellular adenosine pathway in pulmonary infections were published. These include studies elucidating the role of CD73 and A1 in influenza A virus infection (Mycobacterium tuberculosis infection (S. pneumoniae infections (Pseudomonas aeruginosa infection (Around the time that the nfection , 20, thenfection , the rolfections , 22, andnfection . In mostnfection . In conc


Helicobacter pylori (H. pylori) is the most prevalent chronic bacterial infection and is associated with chronic gastritis, peptic ulcer disease, and gastric adenocarcinoma. Although eradication therapy is widely performed for H. pylori infection, adverse events (AEs) are of particular concern in the elderly. This study investigated the efficacy and safety of H. pylori eradication therapy for elderly patients.H. pylori infection was performed to compare clinical indications and outcomes among the younger group (<65 years old), elderly group (65–74 years old), and super-elderly group (>75 years old).Retrospective investigation of 1271 cases  of P < .001), indicating a reluctance for clinicians to treat very old patients, possibly due to unfounded concerns of complications.Chronic gastritis (77.0%) and gastric and/or duodenal ulcer (16.4%) were the most frequent indications for eradication therapy in the cohort. The respective eradication and AE rates for the first and second treatment regimens were 92.1% (1044 of 1133 cases) and 9.1% (103 of 1133 cases) and 84.2% (123 of 146 cases) and 8.9% (13 of 146 cases). No significant differences were detected for eradication rate or AE frequency between the super-elderly group and the other groups. Prior to therapy, the super-elderly group had significantly less frequent chronic gastritis than the other groups but more frequent gastric or duodenal ulcer and post-gastric cancer treatment (all H. pylori eradication is effective and safe, even for elderly patients.Triple therapy for  Conservative estimates suggest that half of the world's population is infected with H. pylori, although country-specific prevalences appear to decline with economic improvement. For example, 70 to 80% of Japanese adults born before 1950 were infected by the bacterium, versus 45% for those born between 1950 and 1960 and 25% for those born between 1960 and 1970. This rapid fall has been attributed to Japan's post-war economic progress and ameliorations in sanitation. However, many elderly patients remain infected with H. pylori until receiving eradication therapy.H. pylori is known to cause chronic gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and gastric mucosa-associated lymphoid tissue (MALT) lymphomas.–6 In addition to symptomatic treatment, H. pylori eradication also appears to reduce the risk of gastric cancer, especially metachronous gastric cancer, and enhance gastric corpus atrophy grade improvement from baseline. Therefore, all individuals with evidence of active H. pylori infection should be offered with eradication therapy in the absence of screening programs for asymptomatic cases.H. pylori eradication therapy have been confirmed as effective and safe. The adverse events (AEs) of eradication therapy are usually mild, with fewer than 10% of patients halting treatment due to unwanted reactions. However, AE rates have been as high as 50% for a particular triple therapy regimen.,12 The frequency of AEs induced by H. pylori eradication therapy in the elderly also remains unclear and possibly misunderstood. This study therefore investigated the efficacy and safety of H. pylori eradication therapy for elderly patients.Several antibiotic regimens for 22.1H. pylori eradication therapy between January 2013 and December 2017 were initially recruited in this retrospective, single-center study. After excluding cases lacking sufficient clinical data for analysis, 1271 patients were ultimately enrolled. The cohort was divided by age into the younger group (≤65 years), elderly group (65–74 years), and super-elderly group (≥75 years) based on the standards of the Japanese national health insurance system for comparisons of clinical characteristics including indication for eradication, eradication regimen, eradication rate, and rate and type of AEs.A total of 1276 patients who had received 2.2H. pylori infection was confirmed by any positive result among serum anti-H. pylori antibodies, urea breath test, stool antigen test, and histopathology. All patients also underwent esophagogastroduodenoscopy (EGD) before eradication therapy to confirm the presence of active chronic gastritis.H. pylori IgG antibody titer was measured using an enzyme immunoassay, with a level of >10 U/mL defined as positive . The urea breath test was performed using infrared spectroscopic analysis according to the manufacturer's instructions . The stool antigen test was carried out using an enzyme-linked immunosorbent assay . Cases were histopathologically defined as H. pylori positive when bacterial bodies were evident in EGD-obtained tissue sections.Serum anti-2.3Patients were initially treated with a regimen of clarithromycin (CAM)-based triple therapy consisting of a proton pump inhibitor (PPI), amoxicillin (AMPC), and CAM for 7 days as a first line treatment. Unresponsive patients identified 4 to 8 weeks later by means of the urea breath test were immediately given PPI, AMPC, and metronidazole (MNZ) as a second regimen. Two-week regimens are currently not approved in Japan. Successful eradication was confirmed by a urea breath test performed 4 weeks or more after completion of triple antibiotic therapy.2.4AEs including diarrhea, eruptions, and constipation Table  were ret2.5U test. Categorical variables were presented as the frequency (percentage) and analyzed using the chi-square test. All statistical tests were two-sided and evaluated at the 0.05 level of significance.Statistical analysis was carried out using StatFlex ver. 7.0.2 . Data were presented as the median ± interquartile range for continuous variables, which were compared using the Mann–Whitney 2.6This investigation was reviewed and approved by the Institutional Review Board of Hokushin General Hospital  . As this was a retrospective observational study, written informed consent was not obtained from participating subjects, although study information was presented in an opt-out format at the hospital. This investigation was conducted in accordance with the principles of the 1975 Declaration of Helsinki as revised in 1983.33.1A flowchart of this study is shown in Fig. 3.2P < .001) but more frequent indications of gastric or duodenal ulcer (both P < .001) and post-gastric cancer treatment (both P < .001). Apart from a significant difference between the younger and elderly groups for first eradication rate (P < .05), no remarkable differences were seen among the groups for the efficacy of either regimen. Moreover, no significant differences were observed in comparisons of AE rates among the groups  based on the age tiers established by the Japanese national health insurance system. Patients with active H. pylori remains unknown, although person-to-person transmission through fecal/oral or oral/oral exposure seems most likely.,14 Person-to-person transmission has been supported by intrafamilial clustering of H. pylori infection, in which infected individuals were more likely to have infected spouses and children than were uninfected individuals.,16 Accordingly, our data highlight the importance of elderly or super-elderly patients who are infected with H. pylori to receive eradication therapy for prevention of cross-infection to younger uninfected family members, especially in multigenerational households.The infection route of  Thus, H. pylori eradication triple therapy along with probiotics might improve both patient treatment and comfort.As expected, the frequency of AEs in our cohort was <10%. The most frequent culprits, diarrhea and constipation, are manageable with drugs and probiotics. It was reported that supplementation with probiotics was effective not only in decreasing eradication therapy-related AEs, but also in increasing eradication rate.This study has several limitations. First, it was conducted as a single-center cohort study of approximately 1000 patients. Secondly, due to its retrospective design, we cannot exclude the possibility that only elderly patients with a favorable systemic condition received eradication therapy.5H. pylori eradication was effective and safe for elderly patients. Further studies are needed to examine the longer-term clinical effects of eradication therapy, such as the prevention of peptic ulcer recurrence, in larger, multi-center, elderly cohorts.Triple therapy for The authors would like to thank Trevor Ralph for his English editorial assistance.Conceptualization: Satoshi Kobayashi.Data curation: Satoshi Kobayashi, Satoru Joshita.Formal analysis: Satoshi Kobayashi.Methodology: Satoshi Kobayashi, Chikara Yamamoto.Project administration: Satoshi Kobayashi, Satoru Joshita.Resources: Satoshi Kobayashi, Chikara Yamamoto, Takumi Yanagisawa, Takayuki Miyazawa, Megumi Miyazawa, Daisuke Kubota, Junichi Sato.Software: Satoshi Kobayashi.Supervision: Eiji Tanaka.Validation: Satoshi Kobayashi, Satoru Joshita, Takeji Umemura.Visualization: Satoshi Kobayashi, Satoru Joshita.Writing – original draft: Satoshi Kobayashi, Satoru Joshita.Writing – review & editing: Chikara Yamamoto, Takeji Umemura, Eiji Tanaka.Satoru Joshita orcid: 0000-0002-6364-9654.


S. mitis is an abundant member of the commensal microbiota of the oral cavity and pharynx, which has the potential to promote systemic infections. By analyzing a collection of S. mitis strains isolated from the oral cavity at commensal states or from systemic infections (blood strains), we established that S. mitis ubiquitously express the surface immunodominant protein, PcsB , required for binding to sucrose-derived exopolysaccharides (EPS). Immuno dot blot assays with anti-PcsB antibodies and RT-qPCR transcription analyses revealed strain-specific profiles of PcsB production associated with diversity in pcsB transcriptional activities. Additionally, blood strains showed significantly higher levels of PcsB expression compared to commensal isolates. Because Streptococcus mutans co-colonizes S. mitis dental biofilms, and secretes glucosyltransferases (GtfB/C/D) for the synthesis of highly insoluble EPS from sucrose, profiles of S. mitis binding to EPS, biofilm formation and evasion of the complement system were assessed in sucrose-containing BHI medium supplemented or not with filter-sterilized S. mutans culture supernatants. These analyses showed significant S. mitis binding to EPS and biofilm formation in the presence of S. mutans supernatants supplemented with sucrose, compared to BHI or BHI-sucrose medium. In addition, these phenotypes were abolished if strains were grown in culture supernatants of a gtfBCD-defective S. mutans mutant. Importantly, GtfB/C/D-associated phenotypes were enhanced in high PcsB-expressing strains, compared to low PcsB producers. Increased PcsB expression was further correlated with increased resistance to deposition of C3b/iC3b of the complement system after exposure to human serum, when strains were previously grown in the presence of S. mutans supernatants. Finally, analyses of PcsB polymorphisms and bioinformatic prediction of epitopes with significant binding to MHC class II alleles revealed that blood isolates harbor PcsB polymorphisms in its functionally conserved CHAP-domain, suggesting antigenic variation. These findings reveal important roles of PcsB in S. mitis-host interactions under commensal and pathogenic states, highlighting the need for studies to elucidate mechanisms regulating PcsB expression in this species. Streptococcus mitis is abundant in multiple oropharyngeal sites, including mucosal and dental surfaces  .surfaces . Althougsurfaces . The broiversity . Howevereumoniae , S. mitis group) . S. mitisurfaces , interacS. mitis strains harbor gene orthologs found in several streptococcal species co-habiting their major host niches , also known as GbpB (Glucan-binding protein B) in S. mutans  of the Piracicaba Dental School, State University of Campinas (UNICAMP), SP, Brazil. These isolates were obtained from the oral mucosal sites  with sterile swabs, using a protocol previously approved by the Ethics Committee of FOP-UNICAMP (protocol 055/2010), as previously described (S. mitis species by using an identification system with specific primers (16S rRNA genes. Eight S. mitis strains with available genomes at the GenBankn = 3) or from the bloodstream of patients with clinical symptoms of bacteremia or septicemia (n = 5) were also analyzed. These strains were provided by Dr. Mogens Killian  and were deposited at CCUG as indicated in 2 in air or aerobic shaking) or anaerobic  atmospheres. Escherichia coli strains TOP10 and BL21 were aerobically grown in Luria-Bertani medium supplemented with ampicillin (100 mg/ml) when required.A total of 20 is study . Twelve escribed . These i primers  and confpcsB were obtained from all the studied strains. Briefly, genomic DNAs were purified using Master Pure DNA purification kit , and applied for amplification of the chromosomal region located 84 bp upstream to 169 bp downstream of pcsB encoding region, using primer sets designed using the genome of strain NCTC12261 as reference   was used as outgroup in the phylogenetic comparisons. Potential peptides with binding affinity to human alleles of Major Histocompatibility Complex (MHC) class II molecules within PcsB sequences were identified using Tepitope/Proped bioinformatics toolComplete sequences of eference . Amplicoeny.fr/) , 2010. OS. mitis PcsB (rPcsB) was obtained by cloning the pcsB encoding region  (A550nm 0.5) after induction of rPcsB expression during 3–4 h with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG). Afterward, rPcsB was purified by affinity chromatography using the Ni-NTA Purification System , as described elsewhere (S. mitis NCTC12261.Recombinant His-tag lsewhere . Samples550nm 0.3). Briefly, adjusted numbers of cells from 18 h cultures in BHI were transferred to 25 ml of fresh BHI medium and incubated (37°C) under aerobiosis (shaking at 160 rpm) or anaerobiosis  until the A550nm 0.3. The culture supernatants of these cultures were collected by centrifugation , neutralized by addition of 1 M NaOH and 10 μM of phenylmethylsulfonyl fluoride (PMSF), and stored at −70°C until use. For preparation of whole cell extracts, the cells harvested from volumes of 25 ml of the same cultures were washed twice with saline solution, suspended in 2 ml of MilliQ water, and mechanically disrupted in a Bead Beater (Biospec Products) with 0.16 g of 0.1-mm zirconia beads (2 cycles of 45 s with 1 min rest on ice). Extracts were then centrifuged  and the supernatants stored at −70°C until use. Culture supernatants were dialyzed (overnight at 4°C) against cold phosphate buffer , and then against cold Tris-HCl . Samples were then 100-fold concentrated by freeze-drying. The protein concentrations of samples were determined using a Bradford assay (Sigma) according to the manufacturer’s protocol.Production of secreted and cell-associated PcsB was analyzed respectively in culture supernatants and whole cell extracts of cells grown in BHI under aerobic and anaerobic conditions until the mid-log phase of growth , under aerobic or anaerobic conditions. Briefly, cells were harvested  from 25 ml of BHI cultures, resuspended in 1 ml of 0.9% saline, and frozen at −70°C. Afterward, these cells were mechanically disrupted in a Mini-BeadBeater (Biospec) with 0.16 g zirconia beads (0.1 mm diameter). RNA was isolated using a modified protocol from the RNeasy Mini kit (Qiagen) described elsewhere  for collection of the supernatants (700 μl), which were then mixed with ethanol (500 μl) and loaded onto columns. Further RNA purification steps were performed as recommended by the manufacturer. Afterward, samples were treated with 10 U Turbo DNase (Ambion) according to the manufacturer’s protocol, for removal of DNA. One microgram of RNA was then used for reverse transcription (RT) with random primers, using the SuperScript III system , as described elsewhere . These three secreted enzymes are required for optimal synthesis of highly stable insoluble glucan EPS from sucrose by S. mutans (gtfBCD mutant obtained in MT8148 (designated BC7s) (S. mutans strains (pH ≈ 6.6–6.8) were supplemented with 1% sucrose and inoculated with adjusted numbers of S. mitis tested strains. Samples were then incubated  for 18 h, and then the intensity of aggregation was scored visually from 0 to 3. To prepare the culture supernatants, the S. mutans strains  were grown in 25 ml of BHI medium  until the mid-log phase of growth . Afterward, these cultures were centrifuged , and the obtained supernatants sterilized by filtration through membranes with pores 0.22 μm in diameter (Corning®). As controls, S. mitis cultures were also mixed with S. mutans culture supernatants not supplemented with sucrose, or with fresh BHI with 1% sucrose.escribed , in BHI . mutans . As conted BC7s)  was alsoS. mutans (MT8148 or BC7s) supplemented with 1% sucrose were inoculated with adjusted numbers of S. mitis cells, and transferred in four replicates (200 μl per well) to polystyrene 96-well plates . After incubation  for 18 h, plates were washed with distilled water to remove loosely attached cells, and the biofilms were stained with crystal violet. Stain was then eluted from biofilms in ethanol (30 min. at room temperature), and the absorbances of the eluates (A575nm) were expressed as indirect measures of biofilm biomass. The planktonic growth (A550nm) was assessed in the same cultures used in the biofilm assays to monitor bacterial growth.Biofilm formation was assessed in microtiter plates as previously described , with moS. mitis strains grown in BHI, BHI 1% sucrose, or in BHI S. mutans UA159 culture supernatant supplemented with 1% sucrose was assessed as previously described , washed twice with PBS (pH 7.4), and incubated  with 20% serum (in PBS). Cells were then washed twice with PBS-Tween 0.05% (PBST), and incubated  with fluorescein isothiocyanate (FITC)-conjugated polyclonal goat anti-human C3 IgG antibody  (1:300 in PBST). Afterward, cells were washed twice with PBST and fixed in 3% paraformaldehyde (in PBS) for analysis on a FACSCalibur flow cytometer (BD Biosciences). Levels of surface-bound C3b on C3b-positive cells were expressed as the geometric mean of fluorescence intensity (MFI), using forward and side scatter parameters to gate at least 25,000 bacteria. Control samples included bacteria treated only with PBS. Heat-inactivated sera (56°C for 20 min) were also used as negative controls in preliminary experiments.Binding of C3b to escribed , with moescribed , 2019, apost hoc Dunn’s multiple comparisons or Mann-Whitney tests. Spearman’s rank correlation was applied to analyze associations between relative amounts of PcsB produced and transcript levels of pcsB. Differences were considered significant when values of p ≤0.05 were obtained.Phenotypic comparisons were performed using Kruskal-Wallis with S. mitis PcsB was initially used to investigate conservation of PcsB orthologs within streptococcal species of the oropharynx. The genomic structures of pcsB loci were then analyzed. PcsB orthologs identified in the analyzed genomes were annotated as SagA (secreted antigen), GSP-781 , CHAP-domain containing protein, or GbpB (Glucan-binding protein B). The degree of protein similarity and gene structure of pcsB loci were compatible with phylogenetic relationships of the species analyzed . Differently in S. oralis strain Uo5, the pcsB downstream genes included a gene encoding an additional “CHAP-domain containing protein,” which was spanned by genes encoding transposases. Upstream to pcsB genes, two genes encoding putative cell-shape determining proteins (mreC/D) were highly conserved in all streptococcal species analyzed, except for S. pyogenes. In Salivarius, Sanguinis, and Mutans group species, as well as in S. pyogenes strain MGAS8232, genes encoding ribose-phosphate diphosphokinase protein (prs) were located downstream to pcsB genes. We could not identify a PcsB ortholog in the S. pyogenes strain M1 GAS SF370. S. mitis PcsB shows high amino acid sequence similarity with most of the oral species , by a leucine zipper domain (amino acids 65–93 in S. mitis), a variable alanine-rich linker region, and a C-terminal CHAP domain  with two conserved residues (Cys315 and His366) involved in peptidoglycan hydrolytic activity  revealed several polymorphisms associated with amino acid changes within conserved functional domains, as well as insertion and deletions at the variable linker region. n = 16) were within the C-terminal extremity of the CHAP-domain (position 305–385). Interestingly, most of the mutations on MHC class II peptides were present in three blood isolates . The blood strain SK569 also accounted for a significant number of amino acid changes (n = 14), but most of them were located within the four N-terminal epitopes. The strains isolated from the oral cavity of infants showed a reduced number of polymorphisms associated with T epitopes (n = 2), and most of the polymorphisms found in these strains were not within functional domains. S. mitis strains, these proteins are more closely related compared to PcsB orthologs expressed by other species of oropharyngeal streptococci.Comparisons of S. mitis strains using protein cell extracts and samples of culture supernatants at mid-log phase of growth. Measures of secreted and cell-associated PcsB were assessed at different atmospheric conditions (aerobiosis and anaerobiosis), because S. mitis colonizes oral niches with varying oxygen tensions. Immuno dot blot analyses of secreted (culture supernatants) and cell extracts revealed significant diversity in total amounts of PcsB produced between strains. Diversity in total amounts of PcsB produced (secreted plus cell-associated PcsB) under aerobic conditions . In both atmospheric conditions, most of the PcsB was present in the culture supernatants  compared to the cell fractions . To further investigate mechanisms underlying diversity in PcsB production, we analyzed pcsB transcriptional activities by RT-qPCR, using RNA samples obtained from cells harvested from the same cultures assayed for protein production. As shown in pcsB in both atmospheric conditions, implying that diversity in PcsB production between strains was promoted by differences in transcriptional activities of the respective pcsB genes.Profiles of PcsB production and protein localization were initially investigated in the studied nditions  was signnditions   and strains isolated from oral sites at commensal states. As shown in n = 4; strains SK569, SK579, SK616, SK1073) and lowest  levels of PcsB (produced under aerobic conditions) to compare bacterial aggregation mediated by sucrose-derived EPS. Of note, different from S. mutans, S. mitis does not secrete glucosyltransferase enzymes (Gtfs) required for the synthesis of insoluble glucan EPS from sucrose  supplemented with sucrose. As controls of Gtf activities, we also supplemented S. mitis cultures with the culture supernatants of the S. mutans gtfBCD triple mutant obtained in MT8148. As shown in S. mutans reference strain UA159 and S. mitis strains show irrelevant aggregation in BHI without sucrose or in S. mutans supernatants in BHI without sucrose, the unique substrate of Gtfs. On the other hand, BHI supplementation with 1% sucrose promoted aggregation of S. mutans UA159, but this phenotype was less evident in most of the S. mitis strains. On the other hand, growth in BHI 1% sucrose medium supplemented with culture supernatants of S. mutans strain MT8148 clearly increased aggregation of S. mitis strains expressing high levels of PcsB, a phenotype not observed in low PcsB-expressing strains . Of note, aggregation of the gtfBCD-defective S. mutans was restored to parent levels, when this defective mutant was grown in BHI-sucrose medium supplemented with MT8148 culture supernatant.In irulence . Thus, w sucrose . Therefo strains . ImportagbpB is required for biofilm formation mediated by sucrose-derived EPS in S. mutans .Because expression of . mutans , and Gbp. mutans , we compernatant . On the ernatant  or BHI-st strain . Consistt strain , but nott strain  or in BHD mutant . Growth S. mutans binding to sucrose-derived EPS promotes the formation of a capsule-like structure for evasion to complement immunity  have resistance to complement deposition compared to low PcsB-producing strains . These comparisons were performed with strains grown either in BHI, BHI 1% sucrose, or in BHI 1% sucrose supplemented with S. mutans UA159 culture supernatants. In all the tested conditions, high PcsB-expressing strains showed significantly lower levels of C3b binding compared to low PcsB producers . The lowest levels of C3b deposition were observed when strains were grown in BHI 1% sucrose culture supernatants of S. mutans UA159. S. mitis strains at conditions that favor the synthesis of sucrose-derived EPS.immunity . We thusS. mitis is an abundant member of the oral microbiota of humans from early life to adulthood  yield similar profiles of pcsB transcription and protein production, further indicating significant influence of the strain background on PcsB expression. These findings highlight the need for studies addressing mechanisms involved in pcsB transcriptional regulation in S. mitis.PcsB/GbpB proteins are essential for bacterial viability in divisome . At leasucan EPS , which aucan EPS . In the S. mutans, S. sanguinis, and S. pneumoniae, gbpB/pcsB are directly regulated by the two-component system (TCS) VicRK  VicRK . In thesS) VicRK . In S. m by CovR , anotherCS CovRS , but in egulator . In S. punctions . Ortholotrain B6 . Thus, aS. mutans account for biofilm enrichment through the synthesis of highly stable EPS  , but not in the presence of culture supernatants of the ΔgtfBCD isogenic S. mutans strain revealed that S. mitis not only interacts with EPS produced by S. mutans-secreted Gtfs, but that strains expressing increased levels of PcsB have enhanced capacity to bind these EPS and to form biofilms. S. mitis binding to EPS synthesized in the presence of S. mutans Gtfs was further associated with reduced levels of C3b deposition.GtfB, GtfC, and GtfD enzymes secreted by inkages) . These einkages) . Differeing Gtfs . This is mutants . Becauseormation , as well. mutans , we hypoS. mitis strains isolated from systemic infections have increased resistance to complement deposition, compared to oral strains   further exemplify how inter-species interactions could promote virulence phenotypes in commensal streptococci with incomplete panels of virulence-associated genes.We have recently reported that  strains , but difrthologs . Capsule evasion , but dif evasion . In addi, gapdh) . AlthougS. mutans GbpB .S. mitis strains from infants, and performed phenotypic and genotypic analyses. EH-C and LA performed protein and transcriptional analyses. LA performed biofilm and complement deposition assays. RM-G, DS, JH, EH-C, and LA analyzed and interpreted the data. RM-G, EH-C, and LA wrote the manuscript. All the authors revised the manuscript and approved its final version.RM-G, DS, EH-C, LA, and WK conceived and designed the experiments. RM-G, EH-C, DS, and WK conceived and performed bioinformatic analyses. EH-C, JT, and MS isolated the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rickettsia spp. in Rhipicephalus sanguineus (sensu lato) collected from free-roaming dogs in 30 communities from five municipalities in the south of Coahuila State, northern Mexico, where Rocky Mountain spotted fever is endemic.The aim of this study was to detect and molecularly identify ompA) gene of Rickettsia spp. were performed.In total, 60 dogs from each municipality were examined for engorged ticks. DNA was isolated from tick pools and conventional PCR assays targeting the 23S-5S ribosomal RNA intergenic spacer and outer membrane protein (n = 1238) were morphologically identified as R. sanguineus (s.l.). Six pools  from four municipalities were positive to Rickettsia spp. DNA sequencing and phylogenetic analyses confirmed the presence of R. rickettsii and R. rhipicephali in R. sanguineus (s.l.) in these ticks.All ticks  from stray dogs in the south of Coahuila. This suggests that stray dogs may play a role in the inter-municipal dissemination of infected ticks in this region. Further research is required to assess whether ticks from stray dogs could serve as good indicators for the molecular xenomonitoring of R. rickettsii in this region. Considering that R. sanguineus (s.l.) is a proven vector of R. rickettsii in Mexico, increased awareness regarding permanent tick control in dogs is warranted.This study confirms the presence of  Rickettsia rickettsii is the causative agent of Rocky Mountain spotted fever (RMSF), a tick-borne disease with increasing incidence rate in North, Central and South American countries, including Mexico [g Mexico –3. RMSF g Mexico . It is wg Mexico .Rhipicephalus sanguineus (sensu lato) [R. rickettsii [R. sanguineus (s.l.) is commonly found infesting both privately-owned and stray dogs in urban and rural areas in Mexicali, the capital of Baja California state, northern Mexico [Recent epidemics of RMSF in south-western USA and northern Mexico have been associated to massive environmental infestations by brown dog ticks, su lato) , 3, 6. Bckettsii , 3, 6, 7ckettsii , 3, 6–8.R. rickettsii-infected brown dog ticks between communities and even between municipalities.In recent outbreaks of RMSF associated with brown dog ticks , most hR. rickettsii in R. sanguineus (s.l.) collected from free-roaming dogs in different municipalities in the south of Coahuila, where a recent outbreak of RMSF was reported.Bearing this in mind, the objective of the present study was to detect and molecularly confirm the presence of R. rickettsii infection rate in ticks is very low, we focused our study on engorged ticks to increase the probability of detection, and also because a previous study showed that 100% of the engorged females became infected upon feeding on dogs with rickettsia [This study was performed from September 2015 to February 2016 in 30 communities belonging to five municipalities  in the south of Coahuila  (Table To assess the circulation of l) Table  and stor23S and 5S ribosomal RNA genes (23S-5S rRNA ITS) of Rickettsia spp., using the primers ITS-F (5′-GAT AGG TCG GGT GTG GAA G-3′) and ITS-R (5′-TCG GGA TGG GAT CGT GTG-3′), as previously described [R. rickettsii-positive sample [DNA samples were initially screened by a conventional PCR assay targeting the internal transcribed spacer (ITS) between the escribed . Brieflye sample  were useompA) gene of Rickettsia spp. belonging to the spotted fever group, using the primers Rr190.70p (5′-ATG GCG AAT ATT TCT CCA AAA-3′) and Rr190.701n (5′-GTT CCG TTA ATG GCA GCA TCT-3′), as described elsewhere [Positive pools were further tested by a conventional PCR assay targeting the outer membrane protein A  software, v.6 [Rickettsia species (using 23S-5S rRNA ITS and ompA) and strains (using ompA) as shown in results. Sequences were aligned with MAFFT (v.7) configured for the highest accuracy using the scoring matrix 200PAM/kD2, alignment strategy MAFFT-FFT-NS-I, gap opening penalty 1.53 and offset value 0.123. The best-fit model of sequence evolution was selected based on corrected Akaike information criterion and Bayesian information criterion implemented in MEGA. The Tamura 3-parameter model [ompA), was chosen for the tree reconstruction. Initial trees for the heuristic search were obtained automatically in MEGA by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using the maximum composite likelihood approach, and then selecting the topology with superior log likelihood value. Reliability of internal branches was assessed using the bootstrapping method with 1000 bootstrap replicates [PCR products were purified with ExoSAP-IT kit , following the manufacturer’s instructions, and purified products sequenced at the Macrogen DNA Sequencing Service . For phylogenetic analysis, maximum likelihood (ML) phylogenetic trees based on are, v.6 . Sequencplicates .The minimum infection rate (MIR) was calculated assuming that each positive pool contained at least one positive tick, using the formula: MIR =  × 100.R. sanguineus (s.l.). In total, 1238 specimens were collected, of which 1063 (85.9%) were engorged females and 175 (14.1%) engorged nymphs. Out of 30 pools tested, six  were found to be positive to Rickettsia spp. DNA . All ticks collected were morphologically identified as R. rickettsii from the USA . Two sequences from San Pedro and one from Torreon showed 99% identity to Rickettsia rhipicephali from south-eastern Brazil (GenBank: KT340606), New Mexico (USA) (GenBank: CP013133) and North Carolina (USA) (GenBank: CP003342). No positive samples were found in the municipality of Matamoros. GenBank accession numbers for the sequences generated in this study are provided in Table By BLAST analysis, the sequences obtained from ticks collected in Francisco I. Madero, Torreon and Viesca presented 99% identity to different sequences of R. rhipicephali and R. rickettsii, which were supported by high bootstrap values  collected from free-roaming dogs in all investigated municipalities except Matamoros. Rickettsia rickettsii was detected in ticks from the municipalities of Francisco I. Madero, Torreon and Viesca. The absence of positive samples from Matamoros is in contrast with a previous study, which reported the presence of R. rickettsii DNA in R. sanguineus (s.l.) [In the present study, we detected the presence of s (s.l.) . Nonethes (s.l.) , 22. In s (s.l.) .R. rhipicephali were obtained, representing the first record of this rickettsial organism in northern Mexico. Rickettsia rhipicephali was originally detected in R. sanguineus (s.l.) in Mississippi, USA [R. rickettsii) has been found in ticks infected with R. rhipicephali, it has been speculated that this rickettsial organism of unknown pathogenicity may protect ticks from subsequent infections with pathogenic rickettsiae [In two pools from Torreon and San Pedro, sequences with 99% of identity to ppi, USA , then inppi, USA . Becausekettsiae .Rickettsia spp. DNA was detected in tick pools, the possibility that this DNA was contained in the dog’s blood ingested by the tick during feeding cannot be ruled out, particularly considering that females with varying degrees of engorgement were included in the pools. Nonetheless, it is interesting to note that the bacteraemia produced by R. rickettsii in dogs is transient, lasting from 3 to 13 days under experimental conditions [R. sanguineus (s.l.) is a recognized vector of R. rickettsii in some epidemic foci of RMSF [Although nditions , 26. The of RMSF , includi of RMSF , where tR sanguineus (s.l.) constitutes a complex of species [Although brown dog ticks are considered to display low affinity for humans , previou species , 12, 32,R. rickettsii transmission. This highlights the need for One Health education initiatives in the RMSF-endemic regions in Mexico, to inform dog owners and the general population about responsible ownership as well as to increase awareness regarding ticks and tick control in dogs. The community-wide application of long-acting tick collars may be effective in controlling focal outbreaks [Even though all dogs enrolled in this study were found free roaming in the streets of the investigated municipalities, most of them are in daily contact with people. In some cases, people from the local communities declared themselves as owners of the dogs and reported that they were typically living outdoors during the day and indoors during the night. This behaviour may contribute to the introduction to ticks into the houses, potentially increasing the risk of utbreaks , but shoutbreaks .R. rickettsii and R. rhipicephali circulating among R. sanguineus (s.l.) collected from free-roaming dogs in Coahuila, where RMSF is endemic. This suggests that free-roaming dogs may play a role in the dissemination of R. rickettsii-infected ticks in this region. Further research is required to assess whether ticks from free-roaming dogs could serve as indicators for the molecular xenomonitoring of R. rickettsii in areas witnessing epidemics of RMSF associated with massive R. sanguineus (s.l.) infestation.The present study confirms the presence of


Patients with knowlesi malaria were older and had lower parasitemia compared to human-only species. Severe knowlesi malaria occurred only in adults. Anemia was common in children despite lower parasitemia. Parasitemia predicted disease severity: Intravenous artesunate is warranted initially for parasitemia>15000/μL. Plasmodium knowlesi is increasingly reported in Southeast Asia, but prospective studies of its clinical spectrum in children and comparison with autochthonous human-only Plasmodium species are lacking.Plasmodium monoinfection presenting to 3 district hospitals in Sabah, Malaysia.Over 3.5 years, we prospectively assessed patients of any age with molecularly–confirmed P < .001) and vivax malaria. In P. knowlesi, World Health Organization–defined anemia was present in 82%  of children vs 36%  of adults. Severe knowlesi malaria occurred in 6.4%  of adults but not in children; the commenst severity criterion was acute kideny injury. No patient had coma. Age, parasitemia, schizont proportion, abdominal pain, and dyspnea were independently associated with severe knowlesi malaria, with parasitemia >15000/μL the best predictor . Two knowlesi-related adult deaths occurred .Of 481 knowlesi, 172 vivax, and 96 falciparum malaria cases enrolled, 44 (9%), 71 (41%), and 31 (32%) children aged ≤12 years. Median parasitemia was lower in knowlesi malaria  than in falciparum (9600/μL; Age distribution and parasitemia differed markedly in knowlesi malaria compared to human-only species, with both uncomplicated and severe disease occurring at low parasitemia. Severe knowlesi malaria occurred only in adults; however, anemia was more common in children despite lower parasitemia. Parasitemia independently predicted knowlesi disease severity: Intravenous artesunate is warranted initially for those with parasitemia >15000/μL. Plasmodium knowlesi human cases in Sarawak, Malaysia, in 2004 [P. knowlesi now accounts for >90% of all government-notified malaria cases [Plasmodium knowlesi is also increasingly reported in areas of western Indonesia [P. knowlesi cases in Southeast Asia threaten regional malaria elimination. Conventional public health measures are unable to target zoonotic transmission to humans from the P. knowlesi reservoir in monkey hosts, particularly outdoors in agricultural or forest areas [Since the initial description of a large focus of zoonotic  in 2004 , knowles in 2004 . In Malaia cases , with >9ia cases . Plasmodndonesia . Difficundonesia  have limndonesia , 12. Desndonesia , increasst areas .Plasmodium falciparum [Prospective studies have described the clinical spectrum of naturally acquired adult knowlesi malaria , 18. Sevlciparum , 19–21.P. falciparum and Plasmodium vivax [P. knowlesi now accounts for around 49% of all reported pediatric malaria cases in Sabah [P. falciparum and P. vivax in district settings.Malaria notification data in knowlesi-endemic areas show a median age of 31 years, much higher than that seen with um vivax , althougum vivax . With thin Sabah . Despitein Sabah , 22, or P. knowlesi or other Plasmodium species infection, and evaluated predictors of disease severity in a coendemic primary care setting.In this study, we compared the predefined clinical spectrum between children and adults with malaria due to 2 and with a total growth-rate adjusted Malaysian census–estimated population in 2016 of 199600 people. Each of the 3 districts in this division has a central referral hospital and subdistrict health clinics, consistent with other districts in Sabah. Malaysian Ministry of Health guidelines stipulate that all patients with fever receive microscopic blood slide screening for malaria parasites, with mandatory hospital admission, free treatment, and notification of positive cases [This study was conducted in Kudat Division, northwest Sabah, Malaysia, covering an area of 4623 kmPlasmodium malariae infection on polymerase chain reaction (PCR), if Plasmodium species PCR was not confirmed, or if cross-check research microscopy was negative. A subset of patients with uncomplicated P. knowlesi and P. vivax malaria was also enrolled in previously reported randomized controlled treatment trials [Patients of all ages presenting to study hospitals with microscopy-diagnosed malaria were enrolled following written informed consent. Children were predefined as age ≤12 years, consistent with Malaysian Ministry of Health pediatric ward admission. Patients were not included in the final analysis if they were pregnant or had t trials .P. knowlesi: hyperparasitemia threshold of 100000/μL, and jaundice defined as bilirubin >50 μmol/L with parasite count >20000/μL and/or creatinine >132 μmol/L [Baseline and longitudinal clinical, laboratory, and epidemiological data were entered using standardized case record forms. Venous blood was taken for baseline investigations and then daily for microscopy and hematology during hospital admission and at the follow-up visit 28 days after treatment initiation. Severe malaria was defined using World Health Organization (WHO) 2014 research criteria , includi2 μmol/L . Nonseve2 μmol/L . AKI wasPlasmodium species confirmation was done using PCR [Microscopic asexual parasite and gametocyte counts were calculated by research microscopists using thick blood smears and quantitated leukocyte count. Standard hospital automated hematology, biochemistry, and microbiology laboratory results were used. Final sing PCR , 30.t test or the Wilcoxon–Mann-Whitney test for 2-group comparisons according to distribution. For categorical variables, χ2 or Fisher’s exact test was used. Logistic regression models were fitted to determine a priori predictors of severe malaria based on standard clinical and laboratory WHO 2014 research criteria [e parasitemia; patients with hyperparasitemia as a sole severity criterion were considered nonsevere.We compared between-group differences with analysis of variance or Kruskal-Wallis testing for continuous variables, and Student criteria  evaluablThis study was approved by the medical research ethics committees of the Ministry of Health, Malaysia; London School of Hygiene and Tropical Medicine, United Kingdom; and Menzies School of Health Research, Australia.P. knowlesi, 172 P. vivax, and 96 P. falciparum malaria cases included in the final analysis. From 2014 to 2015, the estimated minimum yearly malaria incidence in Kudat Division  for P. knowlesi, P. vivax, and P. falciparum was 0.79, 0.40, and 0.19 cases per 1000 people per year, respectively. Patients with knowlesi malaria had a median age of 33 years , higher than those with vivax  and falciparum  malaria . A bimodal age distribution was seen for females with both P. knowlesi and P. falciparum infection. Of P. knowlesi cases, 44 (9%) were children, compared to 71 (41%) of P. vivax cases and 31 (32%) of those with P. falciparum malaria (P < .001). Only 6 (1.3%) knowlesi cases were <5 years of age, with only 1 infant (<1 year), a 6-week-old with no travel history or forest or plantation exposure. Compared to children with P. knowlesi malaria, adults were more likely to be male , with this relationship also evident for P. vivax cases .From October 2012 until April 2016, 811 malaria patients were enrolled . There wP < .001), although vomiting occurred more often in those with P. vivax (P = .033)  and P. falciparum . The highest parasite count recorded for a child with knowlesi malaria was 74365/μL, in an 11-year-old boy with uncomplicated disease. There were 36 (84%) children with knowlesi malaria with nonsevere anemia at presentation, comparable to children with other Plasmodium species infection, with no relationship to parasitemia demonstrated. The lowest hemoglobin level of 5.1 g/dL was seen in a 4-year-old child with knowlesi malaria 2 days after treatment, with 2 other children having minimum hemoglobin levels of 7.0 g/dL, all of whom had a parasite count <1000/μL at presentation. Children with knowlesi malaria had lower neutrophil and lymphocyte counts on presentation compared to those with other Plasmodium species (P = .002). Thirty (68%) children with knowlesi malaria had thrombocytopenia (platelet count <150 × 103/μL), including 4 (9%) with a platelet count <50 × 103 cells/μL. Frequency of thrombocytopenia in children with knowlesi malaria was comparable to those with P. vivax but more common than with P. falciparum malaria . Children with knowlesi malaria were more likely to develop mild to moderate AKI compared to those with P. vivax .Abdominal pain was more common in children with knowlesi compared to vivax malaria  . ChildreP. knowlesi–infected adults was comparable to both children with P. knowlesi and adults with malaria due to other Plasmodium species  or falciparum  malaria , but not P. falciparum or P. vivax infection , and children with knowlesi malaria . As with children, in adult knowlesi malaria parasitemia was not associated with anemia at enrollment after controlling for age. Adults with knowlesi malaria had lower platelet counts than those with other Plasmodium species (P < .001), with thrombocytopenia more common in adults compared to children with knowlesi malaria . The risk of AKI was higher in adult P. knowlesi patients compared to P. vivax , although this did not remain statistically significant after controlling for age; and was also comparable to that seen in both adults with P. falciparum and children with knowlesi malaria. Liver aminotransferases were higher in adults with knowlesi malaria compared to those with P. vivax including after controlling for age (P = .001). Of the 322 adults with knowlesi malaria who had blood cultures, only 1 grew a noncontaminant isolate, a 14-year-old with Neisseria meningitidis.Duration of fever for  species . Adult P malaria . Age wasnfection . Adult PPlasmodium knowlesi was the most common cause of severe malaria, with 28 of 481 (5.8%) knowlesi cases having severe disease (P < .001). The platelet count was lower in severe vs uncomplicated knowlesi malaria , neutrophil count was higher , and proportion of patients with hyponatremia was higher . Five (18%) knowlesi patients had documented hypotension, all of whom had other severity criteria including 2 patients with respiratory distress. Empiric antibiotic treatment was given to 9 (32%) patients with severe knowlesi malaria. Of the 4 severe P. vivax cases, 2 were children with hyperbilirubinemia and a parasite count >20000/μL, both with moderate anemia . The other 2 patients with severe vivax malaria were adults, including a 17-year-old female with severe anemia, and a 53-year-old man with hypotension and respiratory distress. There were 5 patients with severe falciparum malaria (5.2%), including 2 children. No patient with malaria from any Plasmodium species had coma. disease , all of  disease . The mosP = .048). Adults with knowlesi malaria had a higher risk of severe disease compared to adult patients with vivax malaria , and a comparable risk to falciparum malaria.On multivariate logistic regression controlling for age and parasitemia, independent clinical and parasitological predictors of severe disease in knowlesi malaria included schizont proportion >10%, abdominal pain, and dyspnea . Among WP. knowlesi, giving an overall P. knowlesi case fatality risk of 2 of 481 , or 2 of 437  in adults. The P. knowlesi–related deaths were a 62-year-old woman with hyperparasitemia (263772/μL) and moderate AKI (creatinine: 224 μmol/L), who developed hypotension and acute respiratory distress [There were 2 deaths attributed to malaria, both P. knowlesi malaria cases to date, and the first to prospectively compare the clinical spectrum of disease between adults and children. Although 91% of knowlesi malaria cases were adults, morbidity in children was also demonstrated, with an 11-fold higher risk of anemia at presentation and a similar risk of mild to moderate AKI compared to adults [P. knowlesi infection compared with the human-only Plasmodium species may indicate a lower pyrogenic threshold and greater inflammatory response, consistent with poor adaptation of this zoonotic parasite to the human host. Although P. knowlesi has a 24-hour blood-stage life cycle in humans, the low parasitemia in most infections may indicate variable efficiency in human red blood cell (RBC) invasion [This study is the largest series of o adults . The majo adults , 18. Theinvasion . Only a invasion , 18, 32.invasion .P. knowlesi infections were in children compared to those with P. vivax or P. falciparum. The lower incidence of clinical disease from P. knowlesi infection in infants and also older children has been attributed to epidemiological factors such as lower forest exposure [P. vivax or P. falciparum infection, in addition to a lower neutrophil count compared to P. vivax. However, there was a comparable risk of nonsevere anemia and AKI seen in P. knowlesi–infected children as in those with P. vivax or P. falciparum.A lower proportion of exposure , 13, 18,exposure , and asyexposure . Most chexposure . Althougexposure . ChildreP. knowlesi infection was comparable to that seen in P. falciparum. The risk of severe knowlesi malaria in this primary referral setting in Sabah, 6.2% in adults, was similar to district hospital presentations in Sarawak (9.3%) [Plasmodium knowlesi parasitemia [The proportion of adults with severe disease from k (9.3%) , and lowk (9.3%) . Severe k (9.3%) , 18, 32.asitemia  independasitemia . With agasitemia , 36 may P. falciparum, with coma remaining unreported and a lack of the retinal microcirculatory changes found in severe falciparum malaria [P. knowlesi as in P. falciparum infection [Plasmodium transmission may also modulate disease severity.The pathophysiological mechanisms in severe knowlesi malaria are not well understood but likely differ from  malaria . With ennfection , 36, thenfection , requirenfection , howevernfection , requirenfection . Other hPlasmodium species, these management guidelines have contributed to a decline in reported malaria case-fatality rate [Current knowlesi malaria management guidelines in Sabah recommend referral for tertiary care and initial treatment with intravenous artesunate for any patients >50 years of age or with a parasitemia >20000/μL , 40. Aloity rate , 18, 20.P. knowlesi infection causes morbidity at comparatively low parasitemia in both adults and children. Adults are at risk of severe and fatal disease, in contrast to children, among whom this was not demonstrated. A conservative treatment approach utilizing parasite counts to predict severe disease is warranted.In conclusion, although the majority of cases are uncomplicated, Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at Supplementary Table 1Click here for additional data file.


Clostridioides difficile causes more healthcare-associated infections in the United States than any other pathogen, with an estimated 500,000 infections and 29,000 deaths per year [C. difficile first emerged as a healthcare-associated pathogen, infections are increasingly acquired in the community [C. difficile isolates are more diverse than previously presumed and point toward unidentified environmental reservoirs [C. difficile’s epidemiology is essential for future infection prevention efforts, both within healthcare settings and in the community. Here, we provide an overview of the changing epidemiology of C. difficile and current evidence for novel sources of transmission.per year . Althougservoirs . An imprC. difficile first gained attention in the early 2000s, with a rapid increase in healthcare-associated C. difficile infection (CDI) driven by the emergence of the hypervirulent North American pulsed-field gel electrophoresis (PFGE) type 1 (NAP1)/027 (PCR ribotype 027) strain [) strain . High-le) strain ,6. Altho) strain ,7. Over ) strain .For a variety of reasons, the study of CA-CDI is more difficult than healthcare-associated CDI. First, there are few population-based studies of CDI incidence, and the majority of data comes from hospital-based surveillance of CDI among patients admitted to hospitals ,2,9. HosC. difficile, case-control studies consistently associate antibiotic receipt with increased risk of CA-CDI [Similar to healthcare-associated f CA-CDI –12. Protf CA-CDI , 14.C. difficile transmission or if this simply reflects confounding in which chronically ill patients are both at higher risk of C. difficile and more likely to require frequent care.Contact with outpatient healthcare facilities  is also common among patients with CA-CDI . One recRegardless of the exact sources involved, reservoirs for CDI outside of the hospital pose substantial challenges to prevention efforts given that most current initiatives to prevent CDI focus on interventions made within hospitals.C. difficile persists in the environment, is resistant to many routine disinfectants, and causes outbreaks among epidemiologically linked patients. For these reasons, it has been presumed that C. difficile is primarily spread from patient to patient through contaminated hands of healthcare workers, shared equipment, and environmental surfaces. However, current genomic data point toward far more diverse sources of acquisition. A landmark United Kingdom study using whole-genome sequencing found that 45% of C. difficile isolates from hospitalized patients were genetically distinct from all other cases—consistent with a large and extremely diverse reservoir for C. difficile acquisition [uisition .C. difficile transmission have been proposed, few have been rigorously confirmed. The sheer diversity of circulating C. difficile strains challenges efforts to untangle patterns of CDI spread. Aside from revealing much greater diversity than previously anticipated, molecular epidemiologic methods inform many of the potential novel sources for C. difficile transmission, as follows.Although many novel sources for C. difficile strains circulating within the community and among healthcare-acquired cases [C. difficile isolates from a multicenter network and found relatively balanced occurrence of most ribotypes between community and healthcare settings [C. difficile isolates among both community- and healthcare-acquired cases [C. difficile transmission can occur between the healthcare and community environments, existing studies are unable to determine the directionality of transmission. Similarly, even though healthcare contact is frequently associated with CA-CDI, it remains unclear if this reflects patients who are actually at elevated risk because of multiple chronic health problems or if contact with healthcare is truly what is driving the risk.Several large-scale molecular epidemiologic studies have confirmed overlap between ed cases ,15. One settings . A similed cases . The samed cases . DespiteC. difficile carriage rates range from 5.7% to 11.1% among screened inpatients, 7.6% to 24.0% among long-term acute care facility (LTAC) residents, and 16% to 40% among infants  [C. difficile spores in their stool, environmental contamination rates can actually be similar to those with active CDI. Although mathematical modeling studies suggest that implementing contact isolation  for carriers may help to reduce C. difficile transmission rates, clinical data supporting the effectiveness and cost-effectiveness of contact isolation for C. difficile carriers are lacking [disease) . Because lacking . Molecul lacking .Several large-scale cohort and geospatial studies consistently link nursing home or long-term care facility exposure to increased CDI risk . Given hC. difficile contamination across a range of household surfaces even without any known household CDI contacts [Microbiologic surveys of households with a resident family member recently having had CDI confirm widespread contamination. One large-scale case-contact study found an increased risk for CDI among household contacts of patients with CDI for up to 3 months after occurrence of the index infection . Despitecontacts .C. difficile has also been recovered from parks, lawns, soil, beaches, river water, pools, and municipal wastewater, though the relevance of these reservoirs to transmission remains unclear [C. difficile isolates, finding both a high degree of genetic diversity overall but also significant overlap between clinical and environmental isolates [ unclear ,23. One isolates . WhetherC. difficile carriage has been well documented for a wide range of animals, including pets, swine, and cattle [C. difficile isolates from pigs, human CDI cases, and asymptomatically colonized pig farmers. Just under half of paired C. difficile isolates from pig farmers and their respective animals were identical, confirming transmission between animals and humans [d cattle ,25. Ribod cattle . One Dutd humans . Within d humans .C. difficile carriage, based in part on genomic and metabolic data. A point mutation in the trehalose repressor of ribotype 027 increases sensitivity to trehalose concentration, whereas ribotype 078 has acquired a cluster of genes that enhance metabolism of trehalose at low concentrations [C. difficile strains [Which factors are driving the emergence and transmission of particular zoonotic strains has become a particularly active area of interest, currently focused on feed supplements and agricultural antibiotics. Use of trehalose as a feed supplement in swine has been suggested as a risk factor for trations . A recen strains .C. difficile has been reported, including pork, beef, poultry, and processed meats [With high rates of carriage among livestock, it is not surprising that contamination of meat products with ed meats ,32. HoweC. difficile isolates, increasing evidence of transmission outside of the hospital environment, and multiple potential sources of infection challenge current infection control efforts. Improving our understanding of community-acquired C. difficile epidemiology will require detailed prospective collection of wide-ranging exposure-related data on a large scale, coupled with whole-genome sequencing. With the additional issues of widespread outpatient healthcare contact, asymptomatic carriage, and long-term environmental persistence of spores, even the basic distinction between community- versus healthcare-associated CDI may become less relevant with time. Given the challenges posed by current evidence of interspecies transmission and environmental reservoirs of C. difficile, future research in C. difficile prevention will require an integrative multidisciplinary approach, as exemplified by the OneHealth concept.Overall, the high degree of diversity among


Haemaphysalis longicornis salivary cystatin (HlSC-1) and subolesin by RNA interference. Adult unfed female ticks were injected with double-stranded RNA of (A) subolesin, (B) cystatin, (C) subolesin plus cystatin, and (D) injection buffer, then fed alongside normal unfed males up to spontaneous drop-down. The percentage of knockdowns was determined by real-time polymerase chain reaction. Sixty-three percent and 53% knockdown rates were observed in subolesin and cystatin double-stranded RNA-injected ticks respectively, while 32 and 26% knockdown rates of subolesin and cystatin transcript were observed in subolesin plus cystatin double-stranded RNA-injected ticks. Subolesin and/or cystatin knockdown causes a significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Most importantly, combined silencing did not act synergistically, but caused a similarly significant (p < 0.05) reduction in tick engorgement, egg mass weight, and egg conversion ratio. Therefore, the elucidation of multiple antigens may be helpful in the future of vaccines.Currently, multi-antigenic vaccine use is the method of choice for the strategic control of ticks. Therefore, determining the efficacy of combined antigens is a promising avenue of research in the development of anti-tick vaccines. The antigen responsible for blood intake and reproduction has proven suitable as a vaccine antigen. It has been shown to silence  Ceriodaphnia cornuta [The hard tick (Ixodidae) is an obligate, blood-sucking ectoparasite that has gained attention due its role in medicine and veterinary science in the transmission of significant pathogens . As an e cornuta . Althoug cornuta ,7. ThereHaemaphysalis longicornis was reported to transmit a wide variety of pathogens, including Babesia spp., Anaplasma bovis, Theileria spp., Coxiella burnetii, Ehrlichia chaffeensis, spotted fever group rickettsiae, and severe fever with thrombocytopenia syndrome (SFTS) virus throughout China, Korea, and Japan [Among Ixodidae, nd Japan ,12,13,14H. longicornis completes each of its life stages , with the exception of the egg stage, by successive feeding before dropping to molt for the next stage. Tick salivary glands play a vital role in blood engorgement and the transmission of tick-borne pathogens to vertebrates [Blood is the only nutrient that ticks ingest from vertebrates in order to provide themselves with energy to progress through their life stages. As a three-host tick, tebrates . During tebrates . TherefoH. longicornis, three cystatins  have been characterized [H. longicornis, and was found to play a crucial role in the blood-feeding process [Amblyomma americanum [Cystatin, a large, superfamily natural inhibitor of papain-like cysteine proteases, consists of three groups—type 1 (stefin), type 2 (cystatin), and type 3 (kininogen)—which are widely present in vertebrates, invertebrates, and plants, as well as protozoa ,17,18. Tcterized ,22. Amon process . One eleericanum . The othIxodes scapularis [I. scapularis affects fertility in males, and promotes a degeneration of the salivary glands, gut, and reproductive and embryonic tissues in females [A tick protective antigen known as subolesin was recently identified in the apularis . Subolesapularis ,27,28. R females ,29,30,31Rhipicephalus haemaphysaloides has been shown to cause a significant decrease in engorgement rate (5.4%), body weight of engorged ticks (206.15 mg), and egg weight (87.84 mg), in comparison with a control group  [I. scapularis infestations . Additionally, Nijhof et al. [Rhipicephalus microplus resulted in a silencing in both eggs and larvae. The silencing of subolesin expression with RNAi resulted in a reduction in fertility by 93% and 71% in Dermacentor variabilis and I. scapularis, respectively [Injection of subolesin double-stranded RNA (ds RNA) into ctively) . The resf et al.  showed tectively .Boophilus microplus. Two commercial vaccines have been developed for the control of tick infestations in cattle: TickGARD Plus® in Australia, and Gavac® in Cuba. Both vaccines are based on the recombinant antigen Bm86, located on the mid-gut digestive cell. Bm 91 is a low-abundance glycoprotein located in the salivary glands and the midgut of B. microplus . Bm 91 is a homolog of carboxydipeptidase, and shares many biochemical and enzymatic properties with mammalian angiotensin-converting enzymes. An anti-tick vaccine containing multiple antigens is considered more likely to be effective than one containing a single antigen . TherefoIn the present study, using RNAi we silenced salivary subolesin and/or cystatin in order to determine their role in feeding and reproduction. This information may help in selecting multiple antigens as candidate vaccines. H. longicornis (Jeju strain), has been maintained on rabbits at the Laboratory of Veterinary Parasitology, College of Veterinary Medicine and Bio-Safety Research Institute at Chonbuk National University in Iksan, Republic of Korea since 2003 (as described previously) [The hard tick, viously) . The aniH. longicornis were placed on the ears of specific pathogen-free (SPF) New Zealand White rabbits  using a cloth sock attached with tape. After five days of feeding, partially engorged females were removed for salivary gland collection. Ticks were kept for one hour at room temperature for the removal of host tissue. To prevent surface contamination, the ticks were cleaned with distilled water and 70% ethanol. Salivary glands were collected as described elsewhere [Twenty unfed, adult female and male ticks of lsewhere , but witTotal RNA was extracted from the collected salivary glands using a total RNA extraction kit (RiboEx™) in accordance with the manufacturer’s instructions. The concentration of RNA was determined using a NanoDrop™ 2000 spectrophotometer . The sample was then stored at −70 °C. Complementary DNA (cDNA) was synthesized using a transcriptor first-strand cDNA synthesis kit  in accordance with the manufacturer’s instructions, using 1 µg of total RNA and an anchored oligo (dT)18 primer.RT-PCR was performed using BioFACT™ 2X Multi-Star PCR Master Mix with a master cycler gradient . For each gene, cDNA was PCR-amplified using a gene-specific primer . Actin cPCR product was visualized by 1% agarose gel electrophoresis. Then, PCR products were purified using an EZ-Pure™ PCR Purification Kit ver. 2  in accordance with the manufacturer’s instructions. Purified PCR products were sent for sequencing. H. longicornis (HlSC-1) cystatin and subolesin respectively, using an NCBI nucleotide blast search (www.ncbi.nih.gov/blast) (data not shown) (GenBank accession No. EU289292.1).Sequence similarities of 97–100% and 97–98% were observed with The PCR products of subolesin (218 bp) and cystatin (194 bp) were joined to a T7 promoter sequence using T7 promoter-linked (at both the 5′ and 3′ ends) oligonucleotide primers . A T7 pr® 33-gauge needle. After injection, the ticks were kept overnight in a 25 °C incubator with high humidity to observe their survival. The injected ticks of each group were mixed with an equal number of male ticks and then placed on the ears of four SPF rabbits. To observe gene silencing, 10 female ticks of each group were collected after five days of feeding. Their salivary glands were subsequently collected, and real-time PCR was performed for gene expression analysis. The rest of the ticks were fed up to spontaneous drop-down. Feeding time, blood engorgement, egg mass weight, and hatching rate were recorded.Eighty adult, unfed female ticks were divided into four groups, with each group containing an equal number (20) of ticks. The injection dose for the target gene was selected using methods described in previous research ,30. Two ® PrimeScript™ RT-PCR kit II  with a Thermal Cycler Dice™ system . The primer used for gene expression is cited in Ct value, and the percentage of knockdowns was calculated in the same manner as with previous RNAi experiments [Total RNA was collected from the salivary glands of the injected female ticks who had fed for five days, as described above. Real-time PCR was performed to determine the gene expression after knockdown, using a One-step SYBReriments ,39.t-test , executed with Graph Pad Prism 5 . Values were represented in the format of mean ± SE. Values for p that were 0.05 or less were considered to be statistically significant in comparison with the control group.Statistical analysis was performed with a Student’s H. longicornis (HlSC-1) was more abundant in the salivary glands than in the midgut; it plays a crucial role in blood engorgement [The salivary cystatin of orgement . Salivarorgement . CystatiH. longicornis subolesin gene encoding 396 bp  decreased transcript levels (63%) after five days of feeding, in comparison with the control group (p < 0.05) decreased transcript levels (53%), compared with the control group. It was interesting to observe that the cystatin plus subolesin ds RNA-injected group showed significantly (p < 0.05) decreased transcript levels—by 32% and 26% respectively—for subolesin and cystatin  increase in feeding duration (p < 0.05) increased feeding duration  reduced in comparison to the control group (average: 174.13 mg)  B, but nop < 0.05) reduced egg mass (average: 37.25 mg) in comparison with that of the control group (average: 79.87 mg) (p < 0.05), compared with the control group  C. The egol group D. No difp < 0.001) retardation of egg mass and egg conversion ratio (average: 50 mg and 40% respectively), compared with those of the control group, (79.87 mg and 50% respectively)  C,D. No dH. longicornis depends on host blood for survival and reproduction. Tick feeding capability largely depends on its major secretory organ, the salivary glands [Ornithodoros moubata, salivary cystatin OmC2 is an inhibitor of cysteine proteases that resembles human cystin C [A. americanum [A. americanum. Both subolesin and cystatin have significant roles in blood intake. Therefore, we silenced both of these salivary antigens in order to determine their synergistic role in blood intake, as well as their effects on egg production. In our research, it was clear that cystatin knockdown hampers blood engorgement and subsequent egg production. However, the combined effect of subolesin and cystatin silencing has not yet been studied. Our present study aimed to determine the effects of subolesin and/or cystatin knockdown on feeding and reproduction in the H. longicornis.The hard tick y glands . Therefoy glands . The syny glands ,34. Anoty glands . A previcystin C . Therefocystin C . The silericanum . The autR. microplus, I. scapularis, R. haemaphysaloides, Dermacentor marginatus, and D. variabilis [R. haemaphysaloides, indicating their possible future use as part of a cocktail of vaccine antigens [The silencing of the tick protective antigen subolesin by RNAi resulted in a significant reduction of body weight and egg mass, and increased mortality in riabilis ,30,31,32p < 0.05) reduction of engorgement weight in comparison with that of the control group [I. scapularis, A. americanum, and D. marginatus 4D8, ds RNA abrogated egg production [In our present study, the silencing of subolesin by RNAi yielded a significant  reductions of engorgement weight, egg mass, and egg conversion ratio in comparison with the control group were observed. Similarly, 50% knockdown of salivary cystatin caused a significant reduction of tick engorgement in A. americanum [We detected and purified salivary cystatin for the synthesis of ds RNA. After cystatin ds RNA-injected ticks were fed for five days, they showed a reduction of 53% in transcript level. Resultant significant (ericanum . A. americanum, a cystatin and synaptobrevin knockdown reduced successful tick feeding [B. microplus females reportedly led to a significant reduction in tick weight and egg/tick mass, and also to an increased tick mortality [Rhipicephalus sanguineus, silencing of Rs 86 and subA caused a reduction in attachment, engorgement, and oviposition [Amblyomma hebraeum ticks resulted in the silencing of only the subolesin in females which fed together, this was not the case for voraxin [Caenorhabditis elegans, the injection of zfp-1 ds RNA suppressed the lethality produced by mom-2 and hmp-2 ds RNAs [Currently, multi-antigenic vaccines are preferred over single antigenic vaccines ,46. In R feeding . The silortality . In Rhipposition . While t voraxin . However ds RNAs . In our H. longicornis. As multi-antigenic vaccines are more efficient than single-antigenic ones, our findings are expected to lead to subolesin and/or cystatin being considered as vaccine antigens.In the future, combined antigens will be used for controlling ticks and tick-borne diseases. RNAi can be successfully utilized to determine the efficacy of combined gene silencing before vaccination trials. However, it remains a challenge to determine the success of vaccine antigens working together. To our knowledge, the present study is the first to describe the effects of subolesin and/or cystatin knockdown in