• <tr id="yyy80"></tr>
  • <sup id="yyy80"></sup>
  • <tfoot id="yyy80"><noscript id="yyy80"></noscript></tfoot>
  • 99热精品在线国产_美女午夜性视频免费_国产精品国产高清国产av_av欧美777_自拍偷自拍亚洲精品老妇_亚洲熟女精品中文字幕_www日本黄色视频网_国产精品野战在线观看 ?

    Anti-inflammatory and antinociceptive activities of Rhipicephalus microplus saliva

    2018-07-03 08:55:54DFBucciniNunesGGOSilvaONSilvaOLFrancoSEMoreno

    DF. Buccini, ?A. Nunes, GGO. Silva, ON. Silva, OL. Franco,3,4, SE. Moreno?

    1Programa de Pós-Gradua??o em Biotecnologia e Biodiversidade, Universidade Federal do Mato Grosso do Sul, Campo Grande, MS, Brazil

    2S-Inova Biotech Programa de Pós-Gradua??o em Biotecnologia, Universidade Católica Dom Bosco, Campo Grande, MS, Brazil

    3Programa de Pós-Gradua??o em Patologia Molecular, Universidade de Brasília, Brasília, DF, Brazil

    4Centro de Análises Prote?micas e Bioquímicas, Programa de Pós-Gradua??o em Ciências Gen?micas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil

    1. Introduction

    Inflammation is a process involved in protecting the host against injury and infection. It is characterized by redness, swelling, pain as well as tissues and organs dysfunction. Many diseases, including typeⅡ diabetes, cancer, cardiovascular disease and neurodegeneration have recently been considered as possessing a strong inflammatory component[1].

    Molecular processes leading to inflammation are generally related to the activities of cells involved in restoring tissue composition and activity.When cells are exposed to immunological stimulants, proinflammatory cells such as macrophages, neutrophils, monocytes or other host cells may be recruited and initiate to synthesize several molecular mediators that start the inflammation process. Among several biological markers produced in the inflammatory process, the most outstanding are IL-1β, IL-6; IL-8;tumor necrosis factor (TNF-α); nuclear factor-κβ, intercellular adhesion molecule-1, induced cyclooxygenase-2, prostaglandin E2; lipoxygenase (5-LOX); and inducible nitric oxide synthase which stimulates the production of nitric oxide NO[2].

    Inflammation is usually associated with pain as an evolution due to secretion of mediators such as bradykinin, ecoisanoids, histamine,proinflammatory cytokines (TNF, IL-1β and IFN-γ), and chemokines[3,4]. Pain is one of the first symptoms that appear in the process of inflammation; it causes intense suffering and reduces the quality of life. Currently, the therapeutic drugs in pain combat is not totally efficient in terms of efficacy, tolerability and toxicity[4]. Despite the drugs currently available, there is a lack of potential analgesics and anti-inflammatories, as a therapeutic resource for chronic pain[4].Current tools for the treatment of inflammation depend heavily on corticosteroids and AINES, which have several side effects, including osteoporosis, decreased wound healing, ulcerogenic effects and stroke[5].Thus, there is a strong interest in identifying new anti-inflammatory drugs to increase or replace current therapies[5]. Bioprospecting can be an excellent tool to identify and validate new anti-inflammatory targets[6], and ectoparasites could be model organisms to identify new molecules with biotechnological potential[7,8].

    It is already known that tick saliva could be rich in molecules that have anti-inflammatory mechanisms, since such parasites inhibit the host’s inflammatory response, which obtain food successfully during a long period by remaining fixed in cattle. These mechanisms include the inhibition of proteases involved in the inflammatory response,bradykinin hydrolysis by enzymes, binding of salivary proteins to serotonin, leukotriene and histamine[9].

    The large variety of species of ticks already catalogued of molecules present in the saliva of these hematophages make the saliva of these parasites an excellent source for the study of possible pharmacologically active biomolecules. The components present inRhipicephalus microplus(R. microplus) saliva inhibit the host inflammatory response,allowing the parasite to obtain food during long periods[10,11].The impressive ability of tick saliva to modulate host processes demonstrates how we can use these molecules to our advantage.Despite the wide variety of molecules present in saliva that have already been identified, no study has examined the effectsin vivoof saliva as a potential anti-inflammatory and analgesic. Therefore, this study aims to evaluate the antinociceptive and anti-inflammatory potentials as well as thein vivotoxic effects ofR. microplussaliva.

    2. Materials and methods

    2.1. R. microplus saliva collection

    Larvae ofR. micropluswere acquired from engorged females collected in the field (20o23’16.1’’ S 54o36’25.5’’O) and incubated in biochemical oxygen demand (411-D, Nova ética, Brazil) until egg laying.A bovine (Bos taurus) was kept in a closed bay, with a cement floor and free of natural infestations. The bovine was infested with larvae.After 21 d, when the engorged tick females fell, they were collected to obtain the saliva. The experiments were approved by the Dom Bosco Catholic University Committee for Ethics in Animal Experimentation under protocol (No°005/2012). Engorged tick females were washed in sodium hypochlorite 1% and dried gauze. They were injected with 10-20 μL pilocarpine solution 0.2% (Alergan, Brazil) with the aid of a needle measuring 12.7 mm × 0.33 mm (Descarpack). Salivation started after about 10 min and continued for up to 2 h. The saliva was collected using a micropipette (Gilson) and kept on ice. The total saliva obtained was lyophilized (Scientific, VIRTUS, Brazil) and maintained at -80 ℃. The total protein concentration in the saliva was determined by means of the Bradford method[12].

    2.2. Experimental animals

    Adult male albino mice (BALB-c) weighing 22-24 g were used in this study. The animals were obtained from the Central Laboratory for animals of the Dom Bosco Catholic University, Campo Grande, MS,Brazil. The animals were housed in standard sanitized polypropylene cages containing paddy husk as bedding and maintained under controlled conditions of temperature (22 ± 2) ℃ and light-dark cycles(12 h) with free access to standard pellet diet (Nuvilab? CR-1, Nuvital,PR, Brazil) and waterad libitum. The experiments with mice were approved by the Dom Bosco Catholic University Committee for Ethics in Animal Experimentation under protocol (No 005/2012).

    2.3. Hemolytic activity

    The assay for determining hemolytic activity was performed according to the methods of Parket al[13] with minor modifications. Murine erythrocytes were collected from BALB-C mice, washed with 0.9%saline and centrifuged at 580gat 4 ℃ for 2 min. For the experiment,8% blood was used, and distributed in 96-well plate wells, with the addition of different concentrations of crude saliva (300, 200, 100, 50 and 25 μg/mL). The control received Triton X-100 (Vetec) (5 μL diluted in 95 μL of Mili-Q water) and saline 0.9%, and all groups were performed in triplicate. The reading was performed at 540 nm in a microplate reader (Thermo Scientific Multiskan Britain).

    2.4. Cell viability assay

    Neutrophils were obtained from the mice peritoneal cavity, 6 h after intraperitoneal injection of 0.5 mg/animal of carrageenan (Sigma,USA). Animals were euthanized and 3 mL of RPMI medium was injected into the peritoneal cavity (in aseptic conditions); subsequently,exudates were collected and centrifuged at 970gat 10 ℃ for 10 min.Cells were washed twice in sterile PBS and re-suspended in incomplete RPMI medium. Then the cells were counted in a Neubauer chamber.Cell viability was determined by the Tripan Blue exclusion method,and considered adequate when greater than 90%. Cell viability was determined by using the salt dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT-Sigma, USA) colorimetric assay[14,15]. Cell suspension (2×105cells/mL) was plated in a 96-well plate (TPP, Switzerland), incubated with RPMI medium containing 2.5,5, 10, 20 and 40 μg/mL of saliva, and kept in 5% CO2, 37 ℃. Cell viability was evaluated after 24, 48 and 72 h incubation with saliva. After that, all the media were aspirated, and 10 μL of the MTT solution(5 mg/mL) was added to all plate wells and incubated for 4 h under light. After the incubation period, formazan crystals were solubilized by adding the solubilizing solution (isopropyl alcohol and hydrochloric acid-Vetec). The plates were then shaken lightly at room temperature for 5-10 min, so that all the formazan were solubilized and then read at 540 nm on a microplate reader (Thermo Scientific Multiskan Britain).Percentages of cell viability were calculated in relation to untreated cell control.

    2.5. Evaluation of neutrophil migration (NM)

    This assay was performed with crude saliva (10, 15, and 20 mg/kg) 6 h after the administration of the inflammatory stimulus; the groups of animals had been pre-treated or not with compounds with potential anti inflammatory effect. The mice were euthanized in a CO2chamber before starting the experiments of evaluation of NM into the peritoneal cavity.The peritoneal cavity was washed with 3 mL of saline phosphate buffer(Sigma/USA), ethylenediamine tetraacetic acid (Dinamica/Brazil) and PBS/EDTA (5%). Total and differential counts of the cells present were made from the exudate. The total count was performed with the aid of the Neubauer chamber, and the cells were expressed as number of cells×106/mL[16]. The differential counts were performed on slides of the exudate cell smear, stained with (NewProv), and were examined by optical microscope (Eclipse Microscope, Nikon, Japan). One hundred cells were counted per slide, differentiating three cell types: neutrophils,eosinophils and mononuclear cells. The amount of each cell type present in the peritoneal cavity was calculated as the percentage of those cells in counted smears and the total number of cells obtained in the total count.The results were expressed as number of neutrophils (×105/mL)[16,17].

    2.6. Myeloperoxidase activity

    In order to confirm the results obtained in the NM assay, the myeloperoxidase (MPO) activity of neutrophils was measured as described by ALVES-FILHOet al[18]. The peritoneal lavage of the animals treated with saline, carrageenan and 15 mg/kg saliva was subjected to MPO measurement. For this, a solution using tetramethylbenzidine (1.6 mM) and H2O2(0.5 mM) (Thermo Scientific Multiskan Britain) was added to 100 μL of the peritoneal lavage suspension of each animal group, and the change in absorbance at 450 nm was measured. The results were compared to a standard curve performed according to the aforementioned author[18], and expressed as the number of neutrophils (×105/mL).

    2.7. Evaluation of analgesic activity

    2.7.1. Test of abdominal contortions induced by acetic acid

    The analgesic effect was evaluated according to Kosteret al[19]. The animals (BALB-Cn= 5) were pretreated with saliva (15 mg/kg) 15 min prior to pain stimulus with 0.8% acetic acid (10 mL/kgi.p.). The control group received acetylsalicylic acid (Sigma, USA) (AAS – 100 mg/kg). The animals were checked for 30 min after the stimulus and observed for contortions number.

    2.7.2. Formalin test

    The formalin test evaluated the analgesic activity according to the number of licks and/or bites in the paw. BALB-C mice (n= 5) were pretreated with 15 mg/kg of crude saliva, 15 min before the stimulus with 2.5% formalin (30 μL s.p.). Controls received AAS (100 mg/kg). The animals were evaluated in two phases: 1st stage in the first 5 min, taking a 10 min break, and the 2nd phase for the remaining 15 min, observing the number of licks and/or bites in the injected paw[20].

    2.8. Statistical analysis

    The results were expressed as mean ± standard error of the mean(SEM). Significant differences among groups were performed by ANOVA followed by Bonferroni correlation.P< 0.05 was considered to be statistically significant difference. Graphpad Prism? Software(v 5.0; Graph pad Software, USA) was used to perform the statistical analysis.

    3. Results

    Figure 1 demonstrated that saliva had no hemolytic activity in all concentrations tested when compared to the positive control (Triton-X 100).

    Figure 1. Evaluation of hemolytic activity of raw saliva of R. microplus ticks on murine erythrocytes.

    Figure 2. Determination of neutrophil viability (CC) treated with saliva of R.microplus tick for 24 (A); 48 (B) and 72 (C) h.

    Also, in order to evaluate cell viability of neutrophils exposed toR. microplussaliva, the MTT assay was performed. This assay demonstrated that after 24 h of incubation crude saliva was not able to reduce neutrophil (CC) viability at any of the concentrations evaluated(Figure 2). A stimulation of the mitochondrial metabolism was observed at concentrations of 10 μg (51%), 20 μg (86%) and 40 μg(112%) when compared to untreated cells (Figure 2A). After incubation for 48 and 72 h, the saliva was not able to reduce neutrophil viability in any of the concentrations evaluated (Figure 2B and 2C). These results demonstrated thatR. micropluscrude saliva did not present a cytotoxic effect capable of decreasing neutrophil viability. The data suggested that NM decrease into mice peritoneal cavity was not due to the cytotoxic saliva effect over these cells (Figure 3A).

    When we evaluated theR. microplussaliva anti-inflammatory effects over NM to the peritoneal mice cavity, saliva was capable of inhibiting the NM at doses of 15 and 20 mg/kg when compared to the untreated group (carrageenan) (Figure 3A). In order to confirm these data, we evaluated the MPO enzyme as an indicator of neutrophil number. The MPO assay was in agreement with the NM inhibition demonstrated before (Figure 3B). MPO could be used as a local inflammation marker, correlating the enzyme amount to the number of neutrophils present in the tissue. Corroborating with NM data, the group treated with carrageenan presented a higher MPO amount when compared to the groups treated with saliva at the concentration of 15 mg/kg.

    Because pain was a secondary process usually associated with inflammation, we also examined the analgesicR. microplussaliva potential. Figure 4A showed that 15 mg/kg of saliva of theR. microplustick was able to reduce the number of abdominal contortions. The inhibition was 61.62% in the positive control (AAS, 100 mg/kg) and 69.96% in the saliva treated group, when compared to the untreated control; suggesting the remarkable analgesic action of saliva. It was worth noting that the saliva was administered in a concentration almost seven times lower than the AAS and yet it presented a marked analgesic effect.

    Figure 4B demonstrated the evaluation of the analgesic effect ofR.microplussaliva. In this test, two distinct periods of licking activity could be identified, an early phase lasting the first 5 min and a late phase lasting 15 min. The results demonstrated that none of the concentrations used were able to reduce nociception at stage 1,suggesting the absence of central analgesic effects of saliva. However,in phase 2, 15 mg/kg of saliva was able to reduce the nociceptive response triggered by formalin, by 84.41%, and the ASS control was able to inhibit by 76.62% when compared to the untreated group (formalin).Thus, the results showed the analgesic effects ofR. microplussaliva.

    Figure 3. Evaluation of the effect of R. microplus tick saliva on the migration of neutrophils into the peritoneal cavity of mice (A); MPO dosage (B).

    Figure 4. Evaluation of the antinociceptive effect of the saliva of the R.microplus tick by means of the abdominal writhing test (A) and formalininduced lithium test (B) in mice.

    4. Discussion

    R. microplusis one of the bovine parasites of major economic importance, affecting livestock production worldwide and further causing weight loss, reduced milk production, leather quality losses, toxicoses,skin lesions that favor the occurrence of anemia and transmission of pathogens[21]. For the success of parasitism, hematophagous animals need to block host defenses by producing substances such as potent pharmacologic molecules with vasoactive, antihemostatic, antiinflammatory, and immunomodulatory action[10,11] that will be injected together with saliva. The characterization of these substances has revealed a wide variety of compounds with diverse functions, and they are potential sources of pharmacological compounds. Despite many studies, the composition of saliva is not fully understood, nor are its pharmacological effects[9,11].

    According to several studies found in the literature, tick saliva has a number of molecules with varied activities, such as anti-inflammatory and immunosuppressive activity[22], this activity may be due to the lipocalin complex which is determined by the presence of the prostaglandins[23,24], apirases[25] and the lipocalin binding proteins[26].According to Ramachandra and Wikel[27],Dermacentor andersonitick saliva was able to decrease the production of IL-1 and TNF-α by macrophages, IL-2, IFN-γ and by T lymphocytes[27]. Tianet al[28]found in anin vitroexperiment that Amblyomma variegatum saliva inhibited the production of TNF-α, IL-1, CXCL8 and IFN-γ[28].Oliveiraet al[29], demonstrated that the saliva of the tickRhipicephalus sanguineuswas able to inhibit the production of proinflammatory cytokines IL-12 and TNF-α and stimulate IL-10 production by murine dendritic cells,in vitro[29].

    Studies by Tirloneet al[11] have demonstrated thatR. microplussaliva can present a variety of lipocalins. These substances are involved with the immunomodulatory activity of tick saliva[30]. As discussed by Kovar[31], some lipocalins have been characterized as histamine binding proteins, exhibiting anti-inflammatory action[31,32], and presenting as a functional characteristic the ability to act as binders of a large variety of biomolecules, such as nucleotides, bioactive amines (histamine and serotonin), anti-coagulant agents, thromboxanes, leukotrienes,complement system inhibitors and immunoglobulins[22,33].

    Tirloneet al[34,35] also showed that most of the serpinins play crucial roles in managing endopeptidases involved in blood coagulation,fibrinolysis, inflammation and complementary activation[34,35]. It is assumed that the serpin secreted by ticks influences the homeostatic balance of the host to facilitate parasitism[36]. The potential effects of these proteins on host systems have been supported by several studies,showing hematophagous parasite serpins acting as anti-coagulant and anti-inflammatory agents which are essential for successful ectoparasite feeding[30].

    Ribeiro[37] performedin vitrotests with the saliva of theIxodes damminitick, which belongs to the same family asR. microplus[37].Tick saliva contains immunomodulatory compounds that prevent host inflammatory reactions from interfering with the feeding process,creating an environment that allows blood flow without inducing pain[38]. Therefore,R. microplussaliva does have a potential antiinflammatory activity.

    According to the literature, histamine and serotonin secreted by the host at the tick feeding site induce cutaneous inflammation, and ticks must overcome this host response to be successful in feeding[39]. It is suggested thatR. micropluspossesses a blocking agent or neutralizing for histamine and serotonin by decreasing or inhibiting the host’s local immune response[40,41]. The high lipocalin content inR.microplussaliva may also be related to the level required to block the concentration of prostaglandins that accumulate at the feeding site[34,42]. Ticks have been selected during the coevolution process,according to their capacity to disable the host defense responses, with sophisticated mechanisms[10,23].

    In this work it was demonstrated that theR. microplussaliva showed significantin vivoanti-inflammatory and antinociceptive activities. Such activities could be deemed the key elements for its successful parasitic life-style. However, we can take advantage of its biotechnological potential to develop novel anti-inflammatory and analgesic drugs bioinspired by saliva.

    Despite the large number of such drugs available, their side effects and the inefficacy of some drugs under some conditions require a continuous search for new drugs. The data presented here support the development of further studies to elucidate the active principles ofR. microplussaliva and their respective mechanisms of action, and in future to develop novel anti-inflammatory and analgesic drugs.

    Conflict of interest statement

    The authors declare that there is no conflict of interest,

    Acknowledgments

    We thank Souza JVF and Barboza AP for their help in obtaining the saliva and in thein vitroandin vivotests.

    The authors are grateful to FUNDECT (Foundation for Support for the Development of Education, Science and Technology of the State of Mato Grosso do Sul), Coordination for the Improvement of Higher Level Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq) and Foundation for Research Support of the Federal District (FAPDF) for financial support.

    [1] Chen L, Deng H, Cui H, Fang J, Zuo Z, Deng J, et al. Inflammatory responses and inflammation-associated diseases in organs.Oncotarget2018;9(6): 7204-7218.

    [2] Levine TB, Levine AB. Metabolic syndrome and cardiovascular disease.Am Heart J2012; 142(6): 1108-1116.

    [3] Choi JH, Cha DS, Jeon H. Anti-inflammatory and anti-nociceptive properties ofPrunus padus.J Ethnopharmacol2012; 144(2): 379-386.

    [4] Oliveira Júnior JO, Portella Junior CSA, Cohen CP. Inflammatory mediators of neuropathic pain.Revista Dor2016; 17(Suppl 1): S35-42.

    [5] Tamrat Y, Nedi T, Assefa S, Teklehaymanot T, Shibeshi W. Antiinflammatory and analgesic activities of solvent fractions of the leaves ofMoringa stenopetalaBak. (Moringaceae) in mice models.BMC Complement Altern Med2017; 17(1): 473.

    [6] Srilekha V, Krishna G, Seshasrinivas V, Charya MAS. Antibacterial and antiinflammatory activities of marineBrevibacteriumsp.Res Pharm Sci2017;12(4): 283-289.

    [7] Vizioli J, Bulet P, Hoffmann JA, Kafatos FC, Muller HM, Dimopoulos G. Gambicin: A novel immune responsive antimicrobial peptide from the malaria vectorAnopheles gambiae.Proc Natl Acad Sci U S A2001; 98(22):12630-12635.

    [8] Porto WF, Fensterseifer GM, Franco OL.In silicoidentification, structural characterization, and phylogenetic analysis of MdesDEF-2: A novel defensin from the Hessian fly,Mayetiola destructor.J Mol Model2014; 20(7): 2339.

    [9] Esteves E, Maruyama SR, Kawahara R, Fujita A, Martins LA, Righi AA, et al. Analysis of the salivary gland transcriptome of unfed and partially fedAmblyomma sculptumticks and descriptive proteome of the saliva.Front Cell Infect Microbiol2017; 7: 476.

    [10] Ribeiro JM. Role of saliva in blood-feeding by arthropods.AnnuRev Entomol1987; 32: 463-478.

    [11] Tirloni L, Kim TK, Coutinho ML, Ali A, Seixas A, Termignoni C, et al. The putative role ofRhipicephalus microplussalivary serpins in the tick-host relationship.Insect Biochem Mol Biol2016; 71: 12-28.

    [12] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem1976; 72: 248-254.

    [13] Park Y, Kim HN, Park SN, Jang SH, Choi CH, Lim HT, et al. Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12).Biotechnol Lett2004; 26(6): 493-498.

    [14] Takeuchi M, Kobata A. Structures and functional roles of the sugar chains of human erythropoietins.Glycobiology1991; 1(4): 337-346.

    [15] Sieuwerts AM, Klijn JG, Peters HA, Foekens JA. The MTT tetrazolium salt assay scrutinized: How to use this assay reliably to measure metabolic activity of cell culturesin vitrofor the assessment of growth characteristics,IC50-values and cell survival.Eur J Clin Chem Clin Biochem1995; 33(11):813-823.

    [16] Moreno SE, Alves-Filho JC, Alfaya TM, da Silva JS, Ferreira SH, Liew FY.IL-12, but not IL-18, is critical to neutrophil activation and resistance to polymicrobial sepsis induced by cecal ligation and puncture.J Immunol2006; 177(5): 3218-3224.

    [17] Machado RJ, Monteiro NK, Migliolo L, Silva ON, Pinto MF, Oliveira AS, et al. Characterization and pharmacological properties of a novel multifunctional Kunitz inhibitor fromErythrina velutinaseeds.PLoS One2013; 8(5): e63571.

    [18] Alves-Filho JC, de Freitas A, Russo M, Cunha FQ. Toll-like receptor 4 signaling leads to neutrophil migration impairment in polymicrobial sepsis.Crit Care Med2006; 34(2): 461-470.

    [19] Koster R, Anderson M, De-Beer EJ. Acetic acid for analgesic screening.Fed Pro1959; 18(1): 412-418.

    [20] Rosland JH, Hunskaar S, Hole K. The effect of diazepam on nociception in mice.Pharmacol Toxicol1987; 61(2): 111-115.

    [21] Figueiredo A, Fantatto RR, Agnolon IC, Lopes LG, de Oliveira PR, Mathias MIC, et al.In vivostudy of a homeopathic medicine againstRhipicephalus(Boophilus)microplusin dairy cow.Rev Bras Farmacogn2018. Doi:org/10.1016/j.bjp.2018.01.008.

    [22] Anatriello E, Oliveira CJF, Oliveira NB, Fisch A, Milanezi CM, Silva JS, et al. Interaction between saliva’s adenosine and tick parasitism: Effects on feeding and reproduction.Parasite Vector2017; 10(1): 326.

    [23] Ribeiro JM, Makoul GT, Levine J, Robinson DR, Spielman A.Antihemostatic, antiinflammatory, and immunosuppressive properties of the saliva of a tick,Ixodes dammini.J Exp Med1985; 161(2): 332-344.

    [24] Ribeiro JM, Makoul GT, Robinson DR.Ixodes dammini: Evidence for salivary prostacyclin secretion.J Parasitol1988; 74(6): 1068-1069.

    [25] Mans BJ, Gaspar AR, Louw AI, Neitz AW. Apyrase activity and platelet aggregation inhibitors in the tickOrnithodoros savignyi(Acari: Argasidae).Exp Appl Acarol1998; 22(6): 353-366.

    [26] Paesen GC, Adams PL, Harlos K, Nuttall PA, Stuart DI. Tick histaminebinding proteins: Isolation, cloning, and three-dimensional structure.Mol Cell1999; 3(5): 661-671.

    [27] Ramachandra RN, Wikel SK. Modulation of host-immune responses by ticks (Acari: Ixodidae): Effect of salivary gland extracts on host macrophages and lymphocyte cytokine production.J Med Entomol1992; 29(5): 818-826.

    [28] Tian Y, Chen W, Mo G, Chen R, Fang M, Yedid G, et al. An immunosuppressant peptide from the hard tickAmblyomma variegatum.Toxins (Basel)2016; 8(5): 133.

    [29] Oliveira CJ, Cavassani KA, More DD, Garlet GP, Aliberti JC, Silva JS, et al.Tick saliva inhibits the chemotactic function of MIP-1alpha and selectively impairs chemotaxis of immature dendritic cells by down-regulating cellsurface CCR5.Int J Parasitol2008; 38(6): 705-716.

    [30] Blisnick AA, Foulon T, Bonnet SI. Serine protease inhibitors in ticks: An overview of their role in tick biology and tick-borne pathogen transmission.Front Cell Infect Microbiol2017; 7: 199.

    [31] Kovar L. Tick saliva in anti-tick immunity and pathogen transmission.Folia Microbiol (Praha)2004; 49(3): 327-336.

    [32] Paesen GC, Adams PL, Nuttall PA, Stuart DL. Tick histamine-binding proteins: Lipocalins with a second binding cavity.Biochim Biophys Acta2000; 1482(1-2): 92-101.

    [33] Schlehuber S, Skerra A. Lipocalins in drug discovery: From natural ligandbinding proteins to “anticalins”.Drug Discov Today2005; 10(1): 23-33.

    [34] Tirloni L, Reck J, Terra RM, Martins JR, Mulenga A, Sherman NE, et al.Proteomic analysis of cattle tickRhipicephalus (Boophilus) microplussaliva:A comparison between partially and fully engorged females.PLoS One2014;9(4): e94831.

    [35] Tirloni E, Bernardi C, Colombo F, Stella S. Microbiological shelf life at different temperatures and fate ofListeria monocytogenesandEscherichia coliinoculated in unflavored and strawberry yogurts.J Dairy Sci2015;98(7): 4318-4327.

    [36] Syrovets T, Tippler B, Rieks M, Simmet T. Plasmin is a potent and specific chemoattractant for human peripheral monocytes acting via a cyclic guanosine monophosphate-dependent pathway.Blood1997; 89(12): 4574-4583.

    [37] Ribeiro JM. Blood-feeding arthropods: Live syringes or invertebrate pharmacologists?Infect Agents Dis1995; 4(3): 143-152.

    [38] Rodrigues V, Fernandez B, Vercoutere A, Chamayou L, Andersen A, Vigy O. Immunomodulatory effects ofAmblyomma variegatumsaliva on bovine cells: Characterization of cellular responses and identification of molecular determinants.Front Cell Infect Microbiol2017; 7: 521.

    [39] Ribeiro JM, Francischetti IM. Role of arthropod saliva in blood feeding:Sialome and post-sialome perspectives.Annu Rev Entomol2003; 48: 73-88.

    [40] Kemp DH, Bourne A.Boophilus microplus: The effect of histamine on the attachment of cattle-tick larvae--studiesin vivoandin vitro.Parasitology1980; 80(3): 487-496.

    [41] Wikel SK. Host immunity to ticks.Annu Rev Entomol1996; 41: 1-22.

    [42] Tatchell RJ, Bennett GF.Boophilus microplus: Antihistaminic and tranquillizing drugs and cattle resistance.Exp Parasitol1969; 26(3): 369-377.

    18禁黄网站禁片免费观看直播| 精品免费久久久久久久清纯| 国产精品久久久久久精品电影| 男女下面进入的视频免费午夜| 久久99热这里只有精品18| 欧美av亚洲av综合av国产av| 亚洲成人国产一区在线观看| 视频区欧美日本亚洲| 国产精品 国内视频| 亚洲成a人片在线一区二区| 国产日本99.免费观看| 少妇粗大呻吟视频| 亚洲中文日韩欧美视频| 亚洲av电影不卡..在线观看| 成人三级做爰电影| xxxwww97欧美| 欧美日韩一级在线毛片| 亚洲av第一区精品v没综合| 一区福利在线观看| 三级男女做爰猛烈吃奶摸视频| 最新美女视频免费是黄的| www国产在线视频色| 18美女黄网站色大片免费观看| 成人国产一区最新在线观看| 变态另类成人亚洲欧美熟女| 国产伦在线观看视频一区| 亚洲人成网站高清观看| 一本综合久久免费| 老司机深夜福利视频在线观看| 中文资源天堂在线| 日韩欧美在线乱码| 99热这里只有是精品50| 亚洲avbb在线观看| 国产亚洲欧美98| 18美女黄网站色大片免费观看| 黄色视频不卡| 国产av不卡久久| 亚洲av成人av| 亚洲乱码一区二区免费版| 亚洲专区国产一区二区| 国内精品久久久久精免费| 久久久久九九精品影院| 精品国产美女av久久久久小说| 欧美一区二区精品小视频在线| 成人永久免费在线观看视频| 亚洲一区二区三区不卡视频| 女人爽到高潮嗷嗷叫在线视频| 日韩国内少妇激情av| 老熟妇乱子伦视频在线观看| 国产精品一区二区三区四区免费观看 | 999久久久国产精品视频| 波多野结衣高清作品| 亚洲性夜色夜夜综合| 一本久久中文字幕| 两性夫妻黄色片| 成年免费大片在线观看| 国产v大片淫在线免费观看| 国产精品 欧美亚洲| 久久午夜综合久久蜜桃| 夜夜躁狠狠躁天天躁| 怎么达到女性高潮| 麻豆一二三区av精品| 久久 成人 亚洲| 亚洲国产欧美网| 日本熟妇午夜| 精品高清国产在线一区| 欧美日韩瑟瑟在线播放| 欧美日韩精品网址| 欧美激情久久久久久爽电影| 男男h啪啪无遮挡| 欧美高清成人免费视频www| 夜夜躁狠狠躁天天躁| 日日爽夜夜爽网站| 久久国产精品人妻蜜桃| 少妇的丰满在线观看| 一本一本综合久久| 嫁个100分男人电影在线观看| 在线观看免费日韩欧美大片| 中亚洲国语对白在线视频| 亚洲黑人精品在线| 欧美成人性av电影在线观看| 最近视频中文字幕2019在线8| 视频区欧美日本亚洲| 国产精品日韩av在线免费观看| 叶爱在线成人免费视频播放| 亚洲精品粉嫩美女一区| 中文字幕熟女人妻在线| 一个人观看的视频www高清免费观看 | 欧美+亚洲+日韩+国产| 哪里可以看免费的av片| 欧美成人一区二区免费高清观看 | 婷婷丁香在线五月| 亚洲中文日韩欧美视频| 在线观看www视频免费| 男女下面进入的视频免费午夜| 国产精品自产拍在线观看55亚洲| 99在线视频只有这里精品首页| 久久草成人影院| 亚洲在线自拍视频| 国产亚洲av高清不卡| 美女午夜性视频免费| 欧美性猛交╳xxx乱大交人| 在线观看日韩欧美| 久久午夜综合久久蜜桃| 婷婷精品国产亚洲av| 欧美在线黄色| 欧美日韩中文字幕国产精品一区二区三区| 夜夜爽天天搞| 久久久久久亚洲精品国产蜜桃av| 波多野结衣巨乳人妻| 日韩免费av在线播放| 免费av毛片视频| 欧美zozozo另类| 欧美另类亚洲清纯唯美| 国产亚洲精品一区二区www| 男女午夜视频在线观看| 久久香蕉国产精品| 麻豆国产av国片精品| 午夜久久久久精精品| www日本在线高清视频| 日本成人三级电影网站| 人妻夜夜爽99麻豆av| 日本五十路高清| 久久精品夜夜夜夜夜久久蜜豆 | 亚洲一码二码三码区别大吗| 成人手机av| 精品熟女少妇八av免费久了| 欧美丝袜亚洲另类 | 成人高潮视频无遮挡免费网站| 91大片在线观看| 熟女少妇亚洲综合色aaa.| 一本一本综合久久| e午夜精品久久久久久久| 久久久久精品国产欧美久久久| 国产激情偷乱视频一区二区| 99久久99久久久精品蜜桃| 欧美日本视频| 熟妇人妻久久中文字幕3abv| 亚洲国产高清在线一区二区三| 12—13女人毛片做爰片一| 国产精品香港三级国产av潘金莲| 国产黄a三级三级三级人| 国产高清有码在线观看视频 | 18美女黄网站色大片免费观看| 99在线视频只有这里精品首页| 国产亚洲av嫩草精品影院| 91国产中文字幕| 毛片女人毛片| 久久香蕉激情| 别揉我奶头~嗯~啊~动态视频| 中国美女看黄片| 国产伦人伦偷精品视频| 男女午夜视频在线观看| 人人妻,人人澡人人爽秒播| 国产精品久久久久久亚洲av鲁大| 黄色成人免费大全| 熟妇人妻久久中文字幕3abv| 在线国产一区二区在线| 国产精品野战在线观看| 黄色毛片三级朝国网站| 脱女人内裤的视频| 正在播放国产对白刺激| 校园春色视频在线观看| 久久精品国产99精品国产亚洲性色| e午夜精品久久久久久久| 正在播放国产对白刺激| 91在线观看av| 亚洲成人久久性| 久久这里只有精品中国| 男女做爰动态图高潮gif福利片| 丁香欧美五月| 97人妻精品一区二区三区麻豆| 无遮挡黄片免费观看| 亚洲真实伦在线观看| 99re在线观看精品视频| 婷婷六月久久综合丁香| 久久久久久久久久黄片| av有码第一页| 亚洲黑人精品在线| 后天国语完整版免费观看| 狂野欧美白嫩少妇大欣赏| 桃色一区二区三区在线观看| 一卡2卡三卡四卡精品乱码亚洲| 亚洲avbb在线观看| 中文字幕最新亚洲高清| 夜夜躁狠狠躁天天躁| 精品高清国产在线一区| 成人永久免费在线观看视频| www日本在线高清视频| av免费在线观看网站| 久久精品91蜜桃| 午夜福利高清视频| 日本五十路高清| 国产一区二区在线观看日韩 | 亚洲国产欧美人成| 51午夜福利影视在线观看| 久久精品91无色码中文字幕| 国模一区二区三区四区视频 | 亚洲人成77777在线视频| 少妇裸体淫交视频免费看高清 | 亚洲欧美激情综合另类| 老司机福利观看| 两个人看的免费小视频| 一级毛片高清免费大全| 国产伦在线观看视频一区| 又黄又爽又免费观看的视频| 国产精品久久久久久久电影 | 亚洲精华国产精华精| 香蕉国产在线看| 国产视频内射| 丰满人妻熟妇乱又伦精品不卡| 淫妇啪啪啪对白视频| 国产伦一二天堂av在线观看| 777久久人妻少妇嫩草av网站| 国产一级毛片七仙女欲春2| 日韩欧美国产一区二区入口| 丁香欧美五月| 欧美黑人欧美精品刺激| 亚洲狠狠婷婷综合久久图片| 黄频高清免费视频| 一本久久中文字幕| 免费在线观看成人毛片| 久久久久性生活片| 久久久国产成人精品二区| 美女扒开内裤让男人捅视频| 国产精品电影一区二区三区| 国产视频一区二区在线看| 99久久精品热视频| 日韩三级视频一区二区三区| 免费看美女性在线毛片视频| 最新在线观看一区二区三区| 精品国内亚洲2022精品成人| 精品国产亚洲在线| av在线播放免费不卡| 日韩三级视频一区二区三区| 亚洲狠狠婷婷综合久久图片| av视频在线观看入口| 桃红色精品国产亚洲av| 无人区码免费观看不卡| 久久草成人影院| 免费在线观看视频国产中文字幕亚洲| 中文亚洲av片在线观看爽| 精品久久久久久,| 亚洲熟妇熟女久久| 成人午夜高清在线视频| 久久九九热精品免费| 国产久久久一区二区三区| 国产亚洲精品综合一区在线观看 | 视频区欧美日本亚洲| 欧美日韩一级在线毛片| 成人一区二区视频在线观看| 国产爱豆传媒在线观看 | 俺也久久电影网| 精品久久蜜臀av无| 女人爽到高潮嗷嗷叫在线视频| 一二三四在线观看免费中文在| 亚洲真实伦在线观看| АⅤ资源中文在线天堂| 少妇裸体淫交视频免费看高清 | 精品无人区乱码1区二区| 人妻久久中文字幕网| 国产99久久九九免费精品| 亚洲自偷自拍图片 自拍| 午夜福利高清视频| 男女做爰动态图高潮gif福利片| 深夜精品福利| 少妇裸体淫交视频免费看高清 | 免费在线观看成人毛片| 国产精品 欧美亚洲| 久久 成人 亚洲| 老熟妇乱子伦视频在线观看| 一进一出抽搐gif免费好疼| 国产精品久久久av美女十八| 国产三级黄色录像| 国产成人影院久久av| 欧美日韩亚洲国产一区二区在线观看| 日韩精品中文字幕看吧| 亚洲片人在线观看| 国产精品一区二区三区四区久久| 一个人观看的视频www高清免费观看 | 国产伦在线观看视频一区| 国产午夜福利久久久久久| av在线播放免费不卡| 18禁美女被吸乳视频| 久久久久性生活片| 99国产精品99久久久久| 午夜福利在线在线| 国产精品av久久久久免费| 很黄的视频免费| 国产精品电影一区二区三区| 一边摸一边做爽爽视频免费| www日本黄色视频网| 黄色视频不卡| 1024视频免费在线观看| 91麻豆av在线| 日韩欧美在线乱码| 国产一级毛片七仙女欲春2| 免费电影在线观看免费观看| 精品久久久久久久末码| 日本在线视频免费播放| 国内精品一区二区在线观看| 一区福利在线观看| 高清毛片免费观看视频网站| 精品第一国产精品| 午夜福利18| 久久久久九九精品影院| 久久久久久久久中文| 黄色女人牲交| 亚洲色图 男人天堂 中文字幕| 正在播放国产对白刺激| 不卡av一区二区三区| 欧美日本亚洲视频在线播放| 欧美成人性av电影在线观看| 正在播放国产对白刺激| 婷婷精品国产亚洲av| 在线观看午夜福利视频| 成年女人毛片免费观看观看9| 搡老熟女国产l中国老女人| 亚洲全国av大片| 精品乱码久久久久久99久播| 久久性视频一级片| 国产久久久一区二区三区| 欧美乱色亚洲激情| 五月伊人婷婷丁香| 国产成人影院久久av| 色综合婷婷激情| 国产精品综合久久久久久久免费| 黄片小视频在线播放| a级毛片在线看网站| 国产v大片淫在线免费观看| 久久 成人 亚洲| 国产精品一区二区三区四区免费观看 | 国产精华一区二区三区| 成人亚洲精品av一区二区| 亚洲国产日韩欧美精品在线观看 | av有码第一页| 高清毛片免费观看视频网站| 国产久久久一区二区三区| 国产精品一区二区精品视频观看| 五月玫瑰六月丁香| 亚洲人成电影免费在线| 搞女人的毛片| 久久精品aⅴ一区二区三区四区| 日日干狠狠操夜夜爽| e午夜精品久久久久久久| 久久 成人 亚洲| 成人av一区二区三区在线看| 亚洲av成人av| 黄色片一级片一级黄色片| 精品国内亚洲2022精品成人| 精品国产超薄肉色丝袜足j| 久久精品国产亚洲av香蕉五月| 久久亚洲真实| 精品日产1卡2卡| 国产一区二区三区视频了| or卡值多少钱| 亚洲第一电影网av| 亚洲真实伦在线观看| 日韩大码丰满熟妇| 天天一区二区日本电影三级| 天天躁狠狠躁夜夜躁狠狠躁| 国内毛片毛片毛片毛片毛片| 非洲黑人性xxxx精品又粗又长| 免费一级毛片在线播放高清视频| 午夜视频精品福利| 九色成人免费人妻av| 啦啦啦韩国在线观看视频| 国产一区二区三区视频了| 免费电影在线观看免费观看| av视频在线观看入口| 欧美另类亚洲清纯唯美| 亚洲专区字幕在线| 国产三级中文精品| 亚洲国产精品成人综合色| 黄色a级毛片大全视频| 精品人妻1区二区| 久久久久国内视频| 真人做人爱边吃奶动态| 亚洲男人天堂网一区| 精品欧美一区二区三区在线| 91字幕亚洲| 国产成人影院久久av| 淫妇啪啪啪对白视频| 日韩成人在线观看一区二区三区| 国产精品久久久久久精品电影| 国产精华一区二区三区| 午夜免费成人在线视频| 久久99热这里只有精品18| 久久亚洲真实| 最近最新中文字幕大全电影3| 在线观看免费视频日本深夜| 久久天堂一区二区三区四区| 18禁裸乳无遮挡免费网站照片| 蜜桃久久精品国产亚洲av| 免费无遮挡裸体视频| avwww免费| 亚洲五月天丁香| 国产久久久一区二区三区| 国产黄片美女视频| 一区福利在线观看| 色精品久久人妻99蜜桃| 久久热在线av| 国产99白浆流出| 亚洲中文av在线| 免费在线观看成人毛片| 搞女人的毛片| 一进一出好大好爽视频| 老汉色∧v一级毛片| 男女之事视频高清在线观看| 一级毛片女人18水好多| 国产av一区二区精品久久| 亚洲欧美激情综合另类| 亚洲专区国产一区二区| 久久中文字幕人妻熟女| 日韩大尺度精品在线看网址| 国产男靠女视频免费网站| 岛国在线免费视频观看| 两人在一起打扑克的视频| 日韩精品中文字幕看吧| 男女做爰动态图高潮gif福利片| 高清毛片免费观看视频网站| 欧美最黄视频在线播放免费| 亚洲精品一区av在线观看| 久久久久久久精品吃奶| 国产精品野战在线观看| 国产私拍福利视频在线观看| 亚洲一区高清亚洲精品| 999精品在线视频| 国产亚洲精品久久久久5区| 国产精品一区二区免费欧美| 久久精品国产亚洲av香蕉五月| 真人一进一出gif抽搐免费| 成年版毛片免费区| 日韩三级视频一区二区三区| 亚洲欧美精品综合久久99| 国产黄片美女视频| 亚洲,欧美精品.| 亚洲成a人片在线一区二区| 亚洲专区国产一区二区| 欧美日韩亚洲综合一区二区三区_| 国产区一区二久久| 法律面前人人平等表现在哪些方面| 日本五十路高清| 在线观看免费午夜福利视频| 亚洲av五月六月丁香网| 露出奶头的视频| 国产精品一区二区免费欧美| 亚洲国产看品久久| 国产成人av教育| 一边摸一边做爽爽视频免费| 搡老妇女老女人老熟妇| 国产熟女xx| av有码第一页| www.熟女人妻精品国产| 亚洲精品在线观看二区| 国产精华一区二区三区| 亚洲欧美日韩东京热| 亚洲精品美女久久久久99蜜臀| 法律面前人人平等表现在哪些方面| 成人av一区二区三区在线看| 最近最新中文字幕大全免费视频| 成人精品一区二区免费| 日韩欧美国产一区二区入口| 欧美性猛交╳xxx乱大交人| 两个人看的免费小视频| 亚洲真实伦在线观看| 亚洲国产欧美一区二区综合| 欧美黄色淫秽网站| 搡老熟女国产l中国老女人| 男女床上黄色一级片免费看| 久久久精品国产亚洲av高清涩受| 国产午夜精品论理片| videosex国产| 成人一区二区视频在线观看| 少妇被粗大的猛进出69影院| 首页视频小说图片口味搜索| 小说图片视频综合网站| 最近视频中文字幕2019在线8| 国产久久久一区二区三区| 欧美+亚洲+日韩+国产| 这个男人来自地球电影免费观看| 国产亚洲精品综合一区在线观看 | 搡老妇女老女人老熟妇| 最新美女视频免费是黄的| 中文字幕人成人乱码亚洲影| 中国美女看黄片| 又黄又粗又硬又大视频| 国产精品av视频在线免费观看| 可以在线观看的亚洲视频| 国产精品一区二区精品视频观看| 久久中文字幕人妻熟女| 黄色a级毛片大全视频| 丁香欧美五月| x7x7x7水蜜桃| 亚洲中文字幕一区二区三区有码在线看 | 一二三四在线观看免费中文在| 床上黄色一级片| 精品第一国产精品| 午夜影院日韩av| 国产1区2区3区精品| 99热只有精品国产| 国产精品永久免费网站| 精品久久久久久,| 欧美人与性动交α欧美精品济南到| 亚洲av五月六月丁香网| 亚洲中文av在线| 久久久精品大字幕| 国产免费av片在线观看野外av| 中出人妻视频一区二区| 每晚都被弄得嗷嗷叫到高潮| 午夜免费激情av| av免费在线观看网站| 99国产精品一区二区蜜桃av| 不卡av一区二区三区| 日韩欧美免费精品| 亚洲国产欧美网| 欧美性猛交黑人性爽| 99精品在免费线老司机午夜| 99久久精品热视频| 久久伊人香网站| 99热只有精品国产| 亚洲天堂国产精品一区在线| 黄色女人牲交| 国产高清videossex| 午夜免费观看网址| 又爽又黄无遮挡网站| 神马国产精品三级电影在线观看 | 亚洲自偷自拍图片 自拍| 麻豆国产97在线/欧美 | 18禁观看日本| 在线观看日韩欧美| 久久香蕉精品热| 毛片女人毛片| 精品一区二区三区视频在线观看免费| 18美女黄网站色大片免费观看| 国产精品亚洲美女久久久| 日本免费a在线| 在线国产一区二区在线| 长腿黑丝高跟| 欧美久久黑人一区二区| 一a级毛片在线观看| 美女扒开内裤让男人捅视频| 国产精品香港三级国产av潘金莲| 99久久精品国产亚洲精品| 悠悠久久av| 午夜福利在线观看吧| 亚洲自偷自拍图片 自拍| 免费看美女性在线毛片视频| 亚洲av成人一区二区三| 国产一区二区在线av高清观看| 中文字幕久久专区| 久久精品影院6| 看免费av毛片| 变态另类丝袜制服| 高潮久久久久久久久久久不卡| 久久天堂一区二区三区四区| 色av中文字幕| 少妇人妻一区二区三区视频| 狂野欧美白嫩少妇大欣赏| 久久久久久人人人人人| 一本一本综合久久| 久久久久国产精品人妻aⅴ院| 欧美日韩亚洲综合一区二区三区_| 亚洲成av人片在线播放无| 亚洲性夜色夜夜综合| 在线观看舔阴道视频| 香蕉丝袜av| 国产亚洲精品一区二区www| 免费看美女性在线毛片视频| 久久久久免费精品人妻一区二区| a级毛片a级免费在线| 中文字幕久久专区| 国产一区二区在线av高清观看| 波多野结衣巨乳人妻| 麻豆国产97在线/欧美 | 久久精品国产综合久久久| 国产精品自产拍在线观看55亚洲| 啦啦啦观看免费观看视频高清| 国产欧美日韩一区二区三| 国产片内射在线| 草草在线视频免费看| 亚洲av电影不卡..在线观看| 天天躁狠狠躁夜夜躁狠狠躁| 国产日本99.免费观看| 国产精品综合久久久久久久免费| 国产亚洲av嫩草精品影院| 麻豆成人av在线观看| 亚洲国产欧美一区二区综合| 欧美精品啪啪一区二区三区| 丝袜美腿诱惑在线| 亚洲国产日韩欧美精品在线观看 | 亚洲第一电影网av| 国产av一区二区精品久久| 精品日产1卡2卡| 亚洲成人中文字幕在线播放| 熟女少妇亚洲综合色aaa.| 国产一区二区在线观看日韩 | 美女扒开内裤让男人捅视频| 婷婷六月久久综合丁香| 色av中文字幕| 日本 av在线| 精品久久蜜臀av无| 搡老岳熟女国产| 国产单亲对白刺激| 日韩欧美精品v在线| 国产成人欧美在线观看| 波多野结衣高清无吗| 黄色片一级片一级黄色片| 一区二区三区激情视频| 在线观看午夜福利视频| 麻豆成人av在线观看| 这个男人来自地球电影免费观看| 亚洲精品一卡2卡三卡4卡5卡| 国产精品 国内视频| av免费在线观看网站|