저자는 벡터 매개로 질환 동물 리소스 지점 부문 감사드립니다, 특히 안드레아 피터슨, 리사 Massoudi, Verna 오브라이언, 그리고 생쥐와 토끼들의 관심과 유지 보수를위한 존 Liddell. 우리는 또한이 원고 향한 그들의 공헌에 대해 에이미 Ullmann, 테레사 러셀, 그리고 바바라 J. 존슨 감사드립니다. 마지막으로, 우리는이 원고의 촬영과 관련된 모든 위법를 찍는 동안 그래픽 삽화와 주디 Lavelle를 생산 CDC에서 커뮤니케이 션을위한 준회원 이사의 사무실에 Alissa Eckert을 인정하고 싶습니다.

1. 슬라이드 준비 Hemolymph 컬렉션 (동영​​상 1) <ol><li> 부드럽게 표면이 멸균을 위해 10 분간 70 % 에탄올에 나서 5 분 3퍼센트 국소 과산화수소의 동물과 장소에서 적극적으로 먹이 진드기를 제거합니다. </li><li> 자궁 펜은 실란 코팅 현미경 슬라이드에 동그라미를 그려와 자궁 펜 서클 내에서 움직이게 장소. <ol><li> 실란 코팅 슬라이드는 현미경 슬라이드에 hemolymph 최고의 준수를 위해 사용됩니다. </li></ol></li><li> 해부 현미경 (1X 목적, 10X 접안 렌즈, 3.5X 배율)에서 진드기를 볼 수 있습니다. </li><li> 부드럽게 진드기의 다리를 넓히다하고 움직이게 고정하기 위해 집게로 진드​​기의 dorsum 아래로 밀어. </li></ol> 참고 :이 midgut 또는 주사를 움직이게 방해하고 hemolymph을 오염시킬 수 있기를 움직이게 immobilizing가 너무 열심히 누르지 때. <ol start="5"><li> 일시 진드기의 다리 또는 다리를 절단일회용 메스 벌금 포인트와 전자 말초 관절. 감염을 확인하려면 hemolymph 통해 불과 1 다리는 절단해야합니다. 슬라이드에 hemolymph를 수집 몇 다리를 잘라야 수 있습니다. </li></ol> 참고 :이 hemolymph의 midgut 오염을 일으킬 수 있기 때문에 몸 가까이에 다리를 절단하지 마십시오. <ol start="6"><li> 다리 또는 다리가 절단되고 나면 부드럽게 슬라이드를 향해 다리를 밖으로 분비하는 hemolymph 위해 진드기의 dorsum에 압력을 적용하는 것을 계속한다. 부드럽게 hemolymph을 확산하기 위해 슬라이드에 주위를 움직이게 이동합니다. </li></ol> 2. 침샘 제거 (영화 2 및 3) <ol><li> 인산염의 명소 몇 25 μl 수영장 현미경 슬라이드 위에 식염수 (PBS)를 버퍼와 PBS의 수영장 중 하나에 진드기를 놓습니다. </li><li> 해부 현미경 (1X 목적, 10X 접안 렌즈, 3.5X 배율)에서 진드기를 볼 수 있습니다. </li><li> 미세 스쳐 forcep과 진드기를 안정화기준 capitulum (입 부분)이나 진드기의 후면을 개최하여의. </li><li> 진드기의 후면에 벌금 스쳐 포셉를 삽입하고 진드기가 장기를 노출 dorsum있어 최대 썰어. 원하는 경우 midgut이 시간에 제거하고 현미경 슬라이드에 PBS의 신선한 수영장 또는 PBS를 포함 microfuge 튜브로 전송할 수 있습니다. </li><li> 양자 진드기의 다리 옆에 위치한 침샘 (포도 같은 클러스터)의 쌍을 찾아보십시오. 침샘은 진드기의 잔해가 침샘의 파괴와 손실을 줄이기 위해 PBS의 신선한 풀에 여전히 침샘을 포함하는, 주요 진드기 부분을 이동 사이에서 볼 수없는 경우. </li></ol> 참고 : 이전 먹이에 침샘에 오신 것을 나중에 먹이의에 비해 그들로 개발되지 않았기 때문에 찾을 어렵습니다. <ol start="6"><li> 미세 스쳐 포셉와 PBS의 신선한 수영장에서 장소와 진드기의 침샘을 제거합니다. </li><li> 무선좋은 일은 포셉 어떤 외부 미생물과 진드기의 부스러기를 제거하려면이 세차 스텝에게 3-4 번 이상을 반복, 또 다른 깨끗한 25 μl PBS 수영장으로 침샘을 전송 줬지. </li></ol> 참고 : 각각의 침샘의 손실과 혼란을 줄이기 위해 부드럽게 침샘 클러스터를 씻으십시오. <ol start="8"><li> 실란 코팅 슬라이드에 PBS의 깨끗한 수영장으로 또는 PBS를 포함 microfuge 관의 분비를 놓습니다. </li></ol> 3. 침이 수집 (영화 4) <ol><li> 부드럽게 토끼 또는 완전히 부풀어 오른 그들이 내려준 직전 벌금 스쳐 포셉, 약 5~7일 후 첨부 파일을 사용하여 다른 호스트에서 성인 여성의 진드기를 제거합니다. </li><li> 스카치 테이프와 현미경 슬라이드의 한쪽 끝을 향해 거의 부풀어 오른 무슨일을 준수합니다. 테이프는 대략 위치해야 일정한 머리쪽으로 진드기의 dorsum까지 방식 때문에, 진드기의 기준 capitulum (입 부분)가 노출 떠난다.</li><li> 테이프가 5 % pilocarpine 솔루션의 진드기 등 부분의 표면 pipet 5 μl (메탄올에서)의 앞부분은 가장자리를 만나는 곳. 테이프가 pilocarpine은 진드기의 기준 capitulum 접촉 수없이 진드기의 dorsum 이상 pilocarpine을 심하도록 허용합니다. </li><li> 현미경 슬라이드를 향해 무독성 모델링 찰흙 조각 진드기의 mouthparts에서 약 1 인치를 탑재합니다. </li><li> 좋은 스쳐 포셉 원하는 직경으로 뽑았 모세관 28 팁을 끊다 사용합니다. </li><li> 해부 현미경 진드기의 기준 capitulum를 볼 수 있습니다. </li><li> 부드럽게 상악 palps는 모세관 튜브의 외부에있는 수 있도록 뽑아 모세관으로 진드기의 hypostome 맞네요. </li><li> 모세관 장소에서 잡고 모델링 찰흙으로 모세관 튜브의 반대쪽 끝을 누르십시오. </li><li> 높은 습도 (예, 젖은 아빠 줄지어 lidded 스티로폼 상자와 어두운 챔버 내부에 탑재된 침 진드기를 배치<html> 어 수건). 중력이 타액 수집을 도울 수 있도록 hypostome 점 때문에 컨테이너의 하단에 슬라이드를 기울이십시오. </li><li> 상온에서 컨테이너를 배치합니다. </li><li> 밀접하게 1 시간 침 진드기를 모니터링하고, 그것이 파스퇴르의 pipet 벌브와 모세관 튜브 밖으로 추방에 의해 생성되기 때문에 타액을 수집합니다. 첫 번째 시간 후 최소 4 시간 동안 축적 매 시간을 확인합니다. 그것이 생성되기 때문에 타액을 수집하기 위해 계속합니다. </li></ol> 참고 : 충분한 타액이 수행되고있는 연구를 위해 수집되면 타액 수집이 중지됩니다. <ol start="12"><li> 진드기 침되지 않는 이상 타액이 필요할 경우, 타액의 분비는 가끔 hypostome 마사지하는 모세관 튜브를 사용하여 유도된 수있다면. </li><li> 필요한 ° C까지 -80시 타액과 저장소에 테아제 억제제 칵테일의 0.1 볼륨을 추가합니다. </li></ol> 4. 대표 결과 e_content은 &quot;&gt; 영화 1은 부분적으로 싫증이 난 scapularis 요정을 보유하고 현미경 슬라이드에 hemolymph를 수집하기 위해 다리를 절단하는 방법을 보여줍니다. 다리 또는 다리가 맑은 액체가 (그림 1A와 1B) 분비되어 절단되면. 경우 midgut hemolymph 그것이 그대로 흐린 나타나는 절단 다리 (들) (그림 1C 및 1D)로 나오는 파열된다. 요정 48 또는 72 시간 동안 수유 후에도 침샘의 추출이 영화는 2와 3에서 증명된다. 진드기가 파편이 많이 일반적으로이 구멍되면 (기관, malpighian tubules, 혈액 구성되어, 결합 조직 등) 침샘의 손실이나 파괴를 방지하기는 PBS의 신선한 풀에 진드기 이동합니다. 요정이 오픈 컷 후 침샘이있는 그림 2A와 2B 쇼와 그림 2C는 PBS의 수영장에 침샘 클러스터를 제거 보여줍니다. 침이 컬렉션 전에서 설정 scapularis 성인 여성 전<html> s의 영화 4 그림 3. 모세관 튜브에 침 진드기가 영화 5에서 관찰된다. 타액의 분비를 자극하고, 성인 여성 진드기 당 타액의 20 μl를 통해 얻을 수있는 pilocarpine를 사용한 타액 수집이 메서드는. <img alt="1 그림." src="/files/ftp_upload/3894/3894fig1.jpg" /> 그림 1. I.의 scapularis의 요정의 라벨 구조. 영화 1. Ixodes scapularis hemolymph 컬렉션은. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie1.avi">영화를 시청하려면 여기를 클릭하십시오</a> . <img alt="그림 2." src="/files/ftp_upload/3894/3894fig2.jpg" /> 그림 2. 오염 (&amp; B)와 요정의 다리에서 exuding (C &amp; D) hemolymph를 오염. 48시간 먹이기 전의 영화 2. 침샘 추출 scapularis 요정.pload/3894/3894movie2.avi은 &quot;&gt; 영화를보고하려면 여기를 클릭하십시오. 72시간 먹이기 전의 영화 3. 침샘 추출 scapularis 요정. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie3.avi">영화를 시청하려면 여기를 클릭하십시오</a> . <img alt="그림 3." src="/files/ftp_upload/3894/3894fig3.jpg" /> 그림 3. Ixodes scapularis 요정 침샘. (&amp; B)를 사전에 추출에 72시간 수사관 요정의 침샘의 예. (C) 침샘 클러스터를 삭제했습니다. 영화 4. 침이 컬렉션은 성인 전에서 설정 scapularis 여성 눈금. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie4.avi">영화를 시청하려면 여기를 클릭하십시오</a> . <img alt="4 그림." src="/files/ftp_upload/3894/3894fig4.jpg" /> 성인 전에서 그림 4. 타액 수집 scapularis여 ticks. (&amp; B) 모델링 클레이 주최 모세관 튜브의 unpulled 끝에있는 모세관 튜브 뽑아 결국 그 hypostome있는 슬라이드 마운트 틱. (C &amp; D) 침 성인 전을 포함한 Humidified 챔버 scapularis 여성 가고 있습니다. 영화 5. 모세관 튜브에 침 scapularis 여성 무슨일을 Ixodes. <a href="http://www.jove.com/files/ftp_upload/3894/3894movie5.avi">영화를 시청하려면 여기를 클릭하십시오</a> .

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N., Piesman, J., Spielman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+salivary+delivery+of+Lyme+disease+spirochetes+in+vector+ticks+(Acari:+Ixodidae).">Dissemination and salivary delivery of Lyme disease spirochetes in vector ticks (Acari: Ixodidae).</a> J. Med. Entomol. 24, 201-205 (1987).</li><li>Piesman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+Lyme+disease+spirochetes+(Borrelia+burgdorferi.">Transmission of Lyme disease spirochetes (Borrelia burgdorferi.</a> Exp. Appl. Acarol. 7, 71-80 (1989).</li><li>De Silva, A. M., Fikrig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+migration+of+Borrelia+burgdorferi+in+Ixodes+ticks+during+blood+feeding.">Growth and migration of Borrelia burgdorferi in Ixodes ticks during blood feeding.</a> Am. J. Trop. Med. Hyg. 53, 397-404 (1995).</li><li>Horka, H., Cerna-Kyckova, K., Skallova, A., Kopecky, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+saliva+affects+both+proliferation+and+distribution+of+Borrelia+burgdorferi+spirochetes+in+mouse+organs+and+increases+transmission+of+spirochetes+to+ticks.">Tick saliva affects both proliferation and distribution of Borrelia burgdorferi spirochetes in mouse organs and increases transmission of spirochetes to ticks.</a> Int. J. Med. Microbiol. 299, 373-380 (2009).</li><li>Brossard, M., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunology+of+interactions+between+ticks+and+hosts.">Immunology of interactions between ticks and hosts.</a> Med. Vet. Entomol. 11, 270-276 (1997).</li><li>Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+modulation+of+host+immunity:+an+important+factor+in+pathogen+transmission.">Tick modulation of host immunity: an important factor in pathogen transmission.</a> Int. J. Parasitol. 29 (99), 851-859 (1999).</li><li>Binnington, K. C., Kemp, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+tick+salivary+glands+in+feeding+and+disease+transmission.">Role of tick salivary glands in feeding and disease transmission.</a> Adv. Parasitol. 18, 315-339 (1980).</li><li>Guo, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+neutrophil+function+by+two+tick+salivary+proteins.">Inhibition of neutrophil function by two tick salivary proteins.</a> Infect. Immun. 77, 2320-2329 (2009).</li><li>Montgomery, R. R., Lusitani, D., De Boisfleury Chevance, A., Malawista, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tick+saliva+reduces+adherence+and+area+of+human+neutrophils.">Tick saliva reduces adherence and area of human neutrophils.</a> Infect. Immun. 72, 2989-2994 (2004).</li><li>Lima, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+infectivity+of+the+Lyme+disease+spirochete+Borrelia+burgdorferi+derived+from+Ixodes+scapularis+salivary+glands+and+midgut.">Differential infectivity of the Lyme disease spirochete Borrelia burgdorferi derived from Ixodes scapularis salivary glands and midgut.</a> J. Med. Entomol. 42, 506-510 (2005).</li><li>Severinova, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-inoculation+of+Borrelia+afzelii+with+tick+salivary+gland+extract+influences+distribution+of+immunocompetent+cells+in+the+skin+and+lymph+nodes+of+mice.">Co-inoculation of Borrelia afzelii with tick salivary gland extract influences distribution of immunocompetent cells in the skin and lymph nodes of mice.</a> Folia Microbiol. 50, 457-463 (2005).</li><li>Labuda, M., Jones, L. D., Williams, T., Nuttall, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancement+of+tick-borne+encephalitis+virus+transmission+by+tick+salivary+gland+extracts.">Enhancement of tick-borne encephalitis virus transmission by tick salivary gland extracts.</a> Med. Vet. Entomol. 7, 193-196 (1993).</li><li>Kariu, T., Coleman, A. S., Anderson, J. F., Pal, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+Rapid+Transfer+and+Localization+of+Lyme+Disease+Pathogens+Within+the+Tick+Gut.">Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut.</a> J. Vis. Exp. (48), e2544 (2011).</li><li>Edwards, K. T., Goddard, J., Varela-Stokes, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+the+internal+morphology+of+the+Ixodid+tick+Amblyomma+maculatum+koch,+(Acari:Ixodidae);+a+"How-to"+pictorial+dissection+guide.">Examination of the internal morphology of the Ixodid tick Amblyomma maculatum koch, (Acari:Ixodidae); a "How-to" pictorial dissection guide.</a> Midsouth Entomologist. 2, 28-39 (2009).</li><li>Ledin, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Borreliacidal+activity+of+saliva+of+the+tick+Amblyomma+americanum.">Borreliacidal activity of saliva of the tick Amblyomma americanum.</a> Med. Vet. Entomol. 19, 90-95 (2005).</li><li>Ribeiro, J. M., Zeidner, N. S., Ledin, K., Dolan, M. C., Mather, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+much+pilocarpine+contaminates+pilocarpine-induced+tick+saliva?.">How much pilocarpine contaminates pilocarpine-induced tick saliva?.</a> Med. Vet. Entomol. 18, 20-24 (2004).</li><li>Barker, R. W., Burris, E., Sauer, J. R., Hair, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+tick+oral+secretions+obtained+by+three+different+collection+methods.">Composition of tick oral secretions obtained by three different collection methods.</a> J. Med. Entomol. 10, 198-201 (1973).</li><li>Burgdorfer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemolymph+test.+A+technique+for+detection+of+rickettsiae+in+ticks.">Hemolymph test. A technique for detection of rickettsiae in ticks.</a> Am. J. Trop. Med. Hyg. 19, 1010-1014 (1970).</li></ol>

우리는 공개 할게 없다.

진드기의 hemolymph, 침샘, 그리고 타액의 컬렉션 진드기 매개로 병원체 전송 연구에 중요하다, 유행, 보급, 확산, 그리고 진드기 및 호스트 6,11-13,20,23,29 모두에서 끈기. 진드기 30,31 해부하는 방법에는 여러 가지가 있습니다. 그러나 침샘을 수집했을 때 제대로 때문에 침샘이 파열이나 진드기의 유골이 안되는 진드기를 해부하는 것이 중요합니다. 침샘들이 midgut 오염을 제거하고 침?...

<table border="1"><tbody><tr><td> 시약 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> 과산화수소 </td><td> 어부 </td><td> H312-500 </td></tr><tr><td> 에탄올 </td><td> Acros </td><td> 61509-5000 </td></tr><tr><td> PBS </td><td> 보스턴 Bioproducts </td><td> BM-2205 </td></tr><tr><td> 뒤몽 파인 포셉 (3C) </td><td> 어부 </td><td> NC9906085 </td></tr><tr><td> 실란 처리 현미경 슬라이드 </td><td> Bioworld </td><td> 42763007-1 </td></tr><tr><td> 자궁 펜 </td><td> Bioworld </td><td> 21750008-1 </td></tr><tr><td> 슈퍼 서리 플러스 현미경 슬라이드 </td><td> 어부 </td><td> 12-550-18 </td></tr><tr><td> Pilocarpine </td><td> 시그마 </td><td> P6503-5G </td></tr><tr><td> 테아제 억제제 칵테일 </td><td> 시그마 </td><td> P2714 </td></tr><tr><td> # 11 메스 일회용 </td><td> 깃털 </td><td> 2975 # 11 </td></tr><tr><td> 독성 모델링 점토 </td><td&gt; 피셔 </td><td> S17307 </td></tr><tr><td> 모세관 튜브 </td><td> 체이스 과학적 유리, INC </td><td> 40A502 </td></tr></tbody></table>

saliva, salivary gland, and hemolymph collection from ixodes scapularis ticks

진드기는 전세계 찾은 많은 진드기 매개로 질병으로 인간을 괴롭히다하고 있습니다. 진드기 라임 병 및 진드기 매개로 회귀열 (Borrelia SPP.), 록키 마운틴 뇌척수 막염 (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis와 E 동등), anaplasmosis (Anaplasma phagocytophilum), 뇌염 (똑딱똑딱의 원인 병원체에 대한 벡터입니다 매개 뇌염 바이러스), babesiosis (Babesia SPP.), 콜로라도 진드기 열 (Coltivirus) 및 tularemia (Francisella tularensis) 1-8. 제대로 호스트로 전송하기 위해서는 이러한 전염성 요원들은 differentially, 유전자 발현을 조절 진드기 단백질과 상호 작용하고, 진드기 3,9-13을 통해 마이 그 레이션. 예를 들어, 라임 병 에이전트, Borrelia burgdorferi는 진드기의 enzootic 사이클 14,15의 향연과 기근 단계로 차등 유전자 발현을 통해 적응. 또한, Ixodes의 진드기와 같은가를 소비bloodmeal Borrelia은 복제 및 midgut에서 그들이 침샘을 여행하고 퇴학 타액 9,16-19있는 호스트로 전송될 hemocoel,로 마이 그 레이션. 진드기는 피드로서 호스트는 일반적으로 강한 hemostatic와 타고난 면역 반응 11,13,20-22로 응답합니다. 이러한 호스트 반응에도 불구하고, 전 진드기의 타액은 3,11,20,21,23 먹이 진드기들을위한 immunomodulatory있는 단백질, lytic 대리인, anticoagulants 및 fibrinolysins가 포함되어 있기 때문에 scapularis는 몇 일 동안 먹을 수 있습니다. 진드기의 타액이나 침샘 추출물 (SGE)에 의해 점령 immunomodulatory 활동이 많은 진드기 매개로 병원균 3,20,24-27의 전염, 확산 및 보급을 촉진. 더 이상 그것이 진드기 먹이 적극적으로 해부하고 진드기의 타액을 수집하는 데 필수적이며 전염성 요원들이 질병을 일으킬 방법 진드기 매개로 이해하기 위해서. 이 비디오 프로토콜에 대한 해부 기술을 보여줍니다hemolymph의 수집 및 적극적 나도 먹이로부터 침샘의 제거 scapularis nymphs 48 후 72 시간 포스트 마우스 배치. 우리는 또한 성인 여성 I.에서 타액 수집을 보여줍니다 scapularis 눈금.

Watch this Scientific Journal Video about Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks at JoVE.com

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

감염된 진드기의 hemolymph, 침샘, 그리고 타액의 컬렉션 병원균이 질병을 일으킬 방법 진드기 매개로 공부하는 것이 중요합니다. 이 프로토콜에서는 먹이로부터 hemolymph과 침샘을 수집하는 방법을 보여줍니다<em&gt; scapularis을 Ixodes</em&gt; nymphs. 우리는 또한 여성에서 타액 수집을 보여줍니다<em&gt; 전 scapularis</em&gt; 성인.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

saliva-salivary-gland-hemolymph-collection-from-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

29.2K 조회수.  Centers for Disease Control and Prevention. 감염된 진드기의 hemolymph, 침샘, 그리고 타액의 컬렉션 병원균이 질병을 일으킬 방법 진드기 매개로 공부하는 것이 중요합니다. 이 프로토콜에서는 먹이로부터 hemolymph과 침샘을 수집하는 방법을 보여줍니다 scapularis을 Ixodes nymphs. 우리는 또한 여성에서 타액 수집을 보여줍니다 전 scapularis 성인.

비디오: Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

진드기의 RNA 간섭

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

연구

면역학 및 감염병학

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

이종 교잡<em&gt; 현장에서</em&gt; 침샘, 난소, 그리고 벡터 모기의 태아의

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

라임 병 연구의 변속기 및 Xenodiagnosis에 대한 동물에 진드기의 먹이

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

사육<em&gt; Ixodes의 scapularis,</em&gt; 블랙 다리 틱 : 마우스에 미성숙 단계를 먹이

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

쥐에 있는 침 샘 기능의 측정 하는 방법

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

똑바로 Intravital 현미경 Murine Submandibular 침 샘의 준비

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

바이러스로부터 멀리 떨어진 고수율 세포 외 소포 제제의 정제

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

무척 추 병리학 연구를 향한 Rhipicephalus 마이크로 플러스 진드기에 Metarhizium 발 파 포자의 혈 림프 수집 및 접종을 공개

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

마우스 턱밑샘 재생의 실시간 실시간 이미징

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

저자 스포트라이트: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury(부상 후 타액선 재생에서 대식세포-상피세포 상호작용 연구) 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Ex vivo Live Cell Imaging을 통한 타액선의 세포 간 상호 작용 조사

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...