The authors acknowledge the help from the staff of Laboratory Animal Medicine, University of Nevada, Reno. MM received funding from Nevada INBRE.

아래에 설명 된 프로토콜 (수-00682)는 네바다 리노의 대학에서 기관 동물 관리 및 사용위원회 (IACUC)의 승인 네바다, 리노의 동물 연구 윤리위원회의 대학의 지침을 다음입니다. 요약하면, 마우스를 이소 플루 란으로 마취시키고, 노즈콘을 20 ​​분 동안 연속 이소 플루 수준을 유지하기 위해 사용되었다. 수의사의 연고 마취 눈의 건조를 방지하기 위해 사용되었다. 발가락 핀치는 마취 수준을 확인하는 데 사용 된 호흡 속도는 절차를 통해 측정 하였다. 마우스는 각각의 케이지에 보관 의식이 회복 될 때까지 관찰 하였다. 마우스는 틱 공급 한 번만 사용 된 포스트 틱 복구를 안락사시켰다. 안락사는 동물 의학 직원 인원의 연구소에 의해 수행되었다. CO 2 및 자궁 경부 탈구 동물을 안락사에 사용되었다. 참고 : 틱에서 작업이 완료 개인 보호 EQ의 사용을 필요로uipment. 미성숙 틱 카운트 흰색 실험실 코트, 고무 밴드로 캡핑 실험실 코트 슬리브의 개구를 커버하는 긴 니트릴 장갑 막힌 신발을 착용한다. 진드기와 마우스를 감염시키고 들어, 머리 그물, 일회용 작업복, 소매 위에 긴 소매 장갑, 발 커버를 사용합니다. 변덕스러운 진드기를 감지하는 흰색 또는 밝은 색의 작업복을 사용합니다. 정기적으로 진드기를위한 장갑과 소매를 확인합니다. 동물 주택 1. 준비 룸 <ol><li> 진드기가 득실 거리는 집 마우스에 별도의 공간을 지정합니다. 사고 틱 탈출을 방지하기 위해 외부와 도어 안쪽에 끈적 끈적한 매트 또는 양면 카펫 테이프를 놓습니다. </li></ol> 2. 계수 마우스 침입 틱 <ol><li> 모든 7 &quot;× 5&quot;X 14 &quot;플라스틱 용기의 상부 가장자리 주위에 양면 테이프를 넣고 약 2cm 물로 채운다. </li><li> A &amp; 컨테이너의 중앙에 다른 작은 상자 또는 페트리 접시에 놓고 생성 1cm 물로 채우기# 34; 해자 &quot; </li><li> 페트리 접시 안에 애벌레 또는 애벌레가 들어있는 유리 병을 보관하십시오. </li><li> 유리 병에서 애벌레 또는 애벌레를 제거하고 현미경으로 계산하려면 훌륭한 페인트 브러시를 사용하십시오. 50 개의 애벌레 또는 25 명의 애벌레를 별도의 신틸레이션 바이알로 계산하십시오. 나일론 메쉬 나 organdy 천 스크린으로 즉시 튜브를 덮고 고무 밴드로 막으십시오. </li></ol> 3. 미성숙 한 틱으로 생쥐에 감염 <ol><li> 흰색 또는 밝은 색상의 작업대를 사용하고 작업 영역의 둘레에 양면 테이프를 붙입니다. </li><li> isoflurane으로 마우스를 anesthetize. 발가락 핀치로 마취 수준을 확인하십시오. 마취가 끝나면 마우스를 종이 타월로 덮인 가열 패드로 옮기고 계속해서 이소 플루 란 공급을 위해 코 콘에 붙이십시오. </li><li> 건조를 피하기 위해 석유 젤리 기반 안약을 바르십시오. 마우스의 호흡 양상에 따라 isoflurane 수치를 조절하십시오 (분당 80-230 회 호흡은 정상입니다). br 인 경우 isoflurane 수치를 낮추십시오속도 eathing하는) 동물을 죽이는 피하기 분당 호흡 80 미만이다. </li><li> 50 유충 또는 25 요정 중 한 병을 가지고 장소는 페인트 브러시 (그림 1)과 귀 사이의 머리에 모피에서 틱. 진드기 첨부 할 충분한 시간을 제공하기 위해 배치를 다음 20 분 동안 마취 마우스를 유지합니다. </li><li> 정적 뚜껑과 흰색 침구 표준, 플라스틱 신발 상자 형 일회용 마우스 케이지에 마우스를 이동합니다. 일반 마우스 관리에 따라 장난감, 물, 식품 수의를 제공합니다. </li><li> 상단 가장자리 양면 접착 테이프로 줄 지어 큰 쥐 또는 햄스터 케이지 내에서 마우스 케이지를 저장합니다. 물 3cm (도 2)와 외측 케이스를 채운다. </li></ol> 4. 수집은 마우스에서 펜치 <ol><li> 미성숙 틱 3 일 (6) 사이에 급전의 분리. 부풀어 오른 진드기 3 일 후 매일 새장 물 해자를 확인합니다. </li><li> 일 4 6. 고통 사이의 케이지에서 분리 된 진드기를 수집t 브러시 또는 고무 밴드로 고정 나일론 메쉬 천으로 덮인 깨끗한 섬광 유리 병에 부풀어 오른 진드기 및 저장을 따기위한 부드러운 집게. </li><li> 마취 및 나머지 부착 진드기 7 일째에 마우스를 확인하십시오. </li><li> 전술 한 바와 같이 절차에 따라 하루 7 안락사 쥐에 부풀어 오른 진드기 침구, 식품 통, 물병을 확인합니다. 어떤 탈출 unfed 틱을 방지하기 위해 일회용 케이지, 침구, 물 병, 식품 저점을 압력솥. </li></ol> 연방 틱 5. 스토리지 <ol><li> 습도 90 %, 20에서 부풀어 오른 틱 유지 ° C, 및 광 12시 12분 : 습도 및 온도 조절 배양기에서 탈피 암주기가 발생할 때까지. 이 발생하는 약 12 ​​~ 18 주 걸릴 수 있습니다. </li><li> 높은 습도는 진드기의 생존을 촉진하지만, 또한 곰팡이과 성장하는 경향이 눈금을합니다. 연준이 금형을 위해 적어도 일주일에 한 번 현미경 틱 확인합니다. 검출 된 경우, 5, 70 % 에탄올로 세척 생존자분., 물로 씻어 건조 여과지로 전송하고 새로운 깨끗한 바이알로 옮긴다. </li></ol>

<ol>
	<li>Gleim, E. R., 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=Factors+associated+with+tick+bites+and+pathogen+prevalence+in+ticks+parasitizing+humans+in+Georgia,+USA.">Factors associated with tick bites and pathogen prevalence in ticks parasitizing humans in Georgia, USA.</a> Parasites &amp;amp; Vectors. 9 (125), 1-13 (2016).</li><li>Krober, T., Guerin, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+feeding+assays+for+hard+ticks.">In vitro feeding assays for hard ticks.</a> Trends Parasitol. 23 (9), 445-449 (2007).</li><li>Kuhnert, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+of+Hard+Ticks+In+Vitro:+New+Perspectives+for+Rearing+and+for+the+Identification+of+Systemic+Acaricides.">Feeding of Hard Ticks In Vitro: New Perspectives for Rearing and for the Identification of Systemic Acaricides.</a> ALTEX. 13 (2), 76-87 (1996).</li><li>Voigt, W. P., 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=In+vitro+feeding+of+instars+of+the+ixodid+tick+Amblyomma+variegaturn+on+skin+membranes+and+its+application+to+the+transmission+of+Theileria+mutans+and+Cowdria+ruminantium.">In vitro feeding of instars of the ixodid tick Amblyomma variegaturn on skin membranes and its application to the transmission of Theileria mutans and Cowdria ruminantium.</a> Parasitol. 107, 257-263 (1993).</li><li>Gregson, J. D., Smith, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ticks.">Ticks.</a> Insect Colonization and Mass Production. , 49-72 (1966).</li><li>Sonenshine, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biology of Ticks. 2, (1993).</li><li>Bouchard, K. R., Wikel, S. K., Marquaedt, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Care,+maintenance,+and+experimental+infestation+of+ticks+in+the+laboratory+setting.">Care, maintenance, and experimental infestation of ticks in the laboratory setting.</a> Biology of Disease Vectors. , 705-711 (2005).</li><li>Schumaker, T. S., Barros, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+cycle+of+Ornithodoros+(Alectorobius)+talaje.+(Acari:Argasidae)+in+laboratory.">Life cycle of Ornithodoros (Alectorobius) talaje. (Acari:Argasidae) in laboratory.</a> J Med Entomol. 32, 249-254 (1995).</li><li>Banks, C. W., Oliver, J. H., Hopla, C. E., Dotson, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+life+cycle+of+Ixodes+woodi+(Acari:Ixodidae).">Laboratory life cycle of Ixodes woodi (Acari:Ixodidae).</a> J. Med. Entomol. 35, 177-179 (1998).</li><li>James, A. M., Oliver, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+and+host+preference+of+immature+Ixodes+dammini,I.scapularis,and+I.pacificus.(Acari:Ixodidae).">Feeding and host preference of immature Ixodes dammini,I.scapularis,and I.pacificus.(Acari:Ixodidae).</a> J. Med. Entomol. 27, 324-330 (1990).</li></ol>

The authors have nothing to disclose.

프로토콜 내에서 중요한 단계 진드기를 양육하는 것은 우발적 인 탈출을 방지하기 위해 때 안전 조치의 여러 단계를하는 것이 중요합니다. 접착 테이프와 물 해자의 사용은 안전을 보장하는 것이 중요하다. 몸의 온도를 일정하게 유지하기 위해 가열 패드에 마취 된 마우스를 유지하는 것이 중요합니다. 우리는 또한 마우스를 면도하는 진드기 첨부 파일에 대한 추가 혜택을 제?...

진드기는 척추 동물의 가지 의무적 hematophagous 체외 기생충이며, 전 세계적으로 배포됩니다. 미국의 경우, 틱의 적어도 11 종은 공중 보건의 중요성 (1)의 병원균의 벡터이다. Ixodes의 scapularis는 라임 병 (보렐리 burgdorferi에) 열 (B.의 miyamotoi를) 재발, 인간 과립구 anaplasmosis (아나 플라스마의 phagocytophilum), 및 babesiosis의 원인이되는 에이전트로 여러 병원체의 전송에 대한 책임 (Babesia 종.). 실험실에서 연구 야생에서 풍부한에서이 거미를 수집 질병 벡터 등의 I.의 scapularis의 중요성에도 불구하고 항상 가능하지 않습니다. 따라서, 건강한 실험실 사육 틱의 생산 틱 생물학에 대한 연구에 필수적이며, 상호 작용 병원체을 선택합니다. I.의 scapularis을 포함한 모든 하드 진드기 (가족 Ixodidae)의 라이프 사이클은, 계란 세 액티브 구성e 단계 : 유충, 요정, 및 성인. 각 활성 단계는 척추 동물 숙주에게 먹이를줍니다. 첨부와 수유 며칠 동안 진드기와 숙주 사이에서 발생하는 복잡한 상호 작용은 인공 지류를 사용하여 거의 복제 할 수 없으며 대규모 양육 2 , 3 , 4 에 충분한 수의 진드기를 제공하지는 않습니다. 따라서 생쥐와 토끼는 미성숙 (애벌레와 애벌레) 및 진드기의 성숙 단계 (어른)를 키우는 주인으로 가장 많이 사용됩니다. 각 발달 단계에서 혈액 공급을위한 다중 숙주의 요구는 진드기 양육을 복잡하게하고, 시간과 비용을 많이 소비한다 5 , 6 , 7 . 대부분의 진드기 양육 프로토콜은 매달린 와이어 그리드 플로어 케이지 7 , 8 또는 원통형 ca동물이 자유롭게 이동 자체 6, 9, 10을 손질 할 수없는 등의 치수 GE. 이 원통 케이지 후에 와이어 그리드와 콘서트홀 케이지에 전송된다. 충혈, 분리 틱이어서 아래 물로부터 수집된다. 그러나 공급 노출이 Methods는 소변과 곰팡이의 성장을 증가시키고 사망률 (9)을 체크 할 수 배설물로 오염 된 물에 틱. 또한, 눈금 물 통 탈출뿐만 아니라 마우스에 스트레스를 야기 할 가능성을 증가시킨다. 이러한 문제를 회피하기 위해, 우리는 여기에 플라스틱 신발 상자 형 일회용 케이지 내에서 마우스에 애벌레 진드기 먹이를 보여줍니다. 이 방법은 틱 복구 충혈 마우스 증가의 정상적인 동작을 허용하고, 오염물에 의한 진드기 사망률을 감소시킨다.

<table border="0" cellpadding="0" cellspacing="0" width="865">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puralube vet ointment</td>
			<td style="width:207px;">Amazon</td>
			<td style="width:119px;"></td>
			<td style="width:323px;">Available from any Pet store or online store</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Disposable mouse cage</td>
			<td>&#160;Innovive, San Diego, CA&#160;</td>
			<td>MV 2</td>
			<td>Set of bottom and lid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">White Alpha dri bedding&#160;</td>
			<td>Lab Supply, Fort Worth, TX</td>
			<td>&#160;ALPHA-Dri&#8482;</td>
			<td></td>
		</tr>
	</tbody>
</table>

rearing ixodes scapularis, the black-legged tick: feeding immature stages on mice

Ixodes의 scapularis, 라임 병의 벡터, 동부와 중서부 미국에서 가장 중요한 질병 벡터 중 하나입니다. 이 종은 각 개발 단계의 척추 호스트에서 혈액 식사를 필요로하는 세 개의 호스트 틱, 그리고 성인 여성은 재생을위한 혈액 식사를 필요로한다. 수유 5 일 완전히 부풀어 오른 경우 호스트를 내려 - 애벌레는 3 자신의 호스트에 연결 틱. 여러 호스트에이 종속성과 울혈의 긴 부착 시간은 실험실 환경에서 틱 양육을 복잡하게한다. 그러나 상호 작용 틱 생물학을 이해하고 병원체를 체크하기 위해, 건강, 실험실 사육 틱의 생산이 필수적이다. 여기, 우리가 쥐에 먹이 미성숙 한 진드기에 대한 간단하고 비용 효율적인 프로토콜을 보여줍니다. 우리는 생쥐에서 감소 스트레스에 대한 기존의 프로토콜을 수정 틱 위스콘신의 접촉을 피하기 위해 메쉬 바닥없이 일회용 케이지를 사용하여 성공과 생존을 공급 틱 증가생쥐 소변과 대변으로 오염 된 물.

마우스 숙주의 먹이 효율을 높이고 스트레스를 줄이기 위해 기존의 진드기 양육 프로토콜 6 , 10 을 수정했습니다. 결과는 표준 슈 박스 스타일 마우스 케이지가 진드기 양육에 적합 함을 보여줍니다. 화이트 침구는 깍지 줍기를 쉽게 수집 할 수 있도록 좋은 대비를 제공했습니다. 대부분의 진드기는 수유 후 용기의 벽을 올라가?...

Watch this Scientific Journal Video about Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice at JoVE.com

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

건강한 실험실에서 재배 한 진드기의 생산은 진드기 생물학 및 진드기 - 병원체 상호 작용에 대한 연구에 필수적입니다. 여기서 우리는 마우스에 비용 효과적이고 스트레스가 적은 미성숙 진드기 먹이를위한 간단한 프로토콜을 시연합니다.

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 ...

rearing-ixodes-scapularis-black-legged-tick-feeding-immature-stages

Research

JoVE Journal

Immunology and Infection

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

11.9K 조회수.  University of Nevada, Reno.  건강한 실험실에서 재배 한 진드기의 생산은 진드기 생물학 및 진드기 - 병원체 상호 작용에 대한 연구에 필수적입니다. 여기서 우리는 마우스에 비용 효과적이고 스트레스가 적은 미성숙 진드기 먹이를위한 간단한 프로토콜을 시연합니다. 

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

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

틱 굿 내에서 빠른 전송과 라임 질병 병원균의 현지화을위한 방법

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

연구

면역학 및 감염병학

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

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

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

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

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 ...

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and tomato. The susceptibility of M. sexta larvae to a variety of entomopathogenic bacterial species1-5, as well as the wealth of information available regarding the insect's immune system6-8, and the pending genome sequence9 make it a good model organism for use in studying host-microbe interactions during pathogenesis. In addition, M. sexta larvae are relatively large and easy to manipulate and maintain in the laboratory relative to other susceptible insect species. Their large size also facilitates efficient tissue/hemolymph extraction for analysis of the host response to infection.
The method presented here describes the direct injection of bacteria into the hemocoel (blood cavity) of M. sexta larvae. This approach can be used to analyze and compare the virulence characteristics of various bacterial species, strains, or mutants by simply monitoring the time to insect death after injection. This method was developed to study the pathogenicity of Xenorhabdus and Photorhabdus species, which typically associate with nematode vectors as a means to gain entry into the insect. Entomopathogenic nematodes typically infect larvae via natural digestive or respiratory openings, and release their symbiotic bacterial contents into the insect hemolymph (blood) shortly thereafter10. The injection method described here bypasses the need for a nematode vector, thus uncoupling the effects of bacteria and nematode on the insect. This method allows for accurate enumeration of infectious material (cells or protein) within the inoculum, which is not possible using other existing methods for analyzing entomopathogenesis, including nicking11 and oral toxicity assays12. Also, oral toxicity assays address the virulence of secreted toxins introduced into the digestive system of larvae, whereas the direct injection method addresses the virulence of whole-cell inocula.
The utility of the direct injection method as described here is to analyze bacterial pathogenesis by monitoring insect mortality. However, this method can easily be expanded for use in studying the effects of infection on the M. sexta immune system. The insect responds to infection via both humoral and cellular responses. The humoral response includes recognition of bacterial-associated patterns and subsequent production of various antimicrobial peptides7; the expression of genes encoding these peptides can be monitored subsequent to direct infection via RNA extraction and quantitative PCR13. The cellular response to infection involves nodulation, encapsulation, and phagocytosis of infectious agents by hemocytes6. To analyze these responses, injected insects can be dissected and visualized by microscopy13, 14.

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

The method described here utilizes direct injection of entomopathogenic bacteria into the hemocoel of Manduca sexta insect larvae. M. sexta is a commercially available and well-studied insect. Thus, this method represents a simple approach to analyzing host-bacterial interactions from the perspective of one or both partners.

의 양육 및 분사<em&gt; Manduca sexta</em박테리아 독성을 평가하기&gt; 애벌레

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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 ...

Measuring Local Anaphylaxis in Mice

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. Exposure to an allergen in a sensitized individual can result in clinical symptoms that vary from minor erythema to life threatening anaphylaxis. In the laboratory, various animal models have been developed to understand the mechanisms driving allergic responses. Herein, we describe a detailed method for measuring changes in vascular permeability to quantify localized allergic responses. The local anaphylaxis assay was first reported in the 1920s, and has been adapted from the technique published by Kojima et al. in 20071. In this assay, mice sensitized to OVA are challenged in the left ear with vehicle and in the right ear with OVA. This is followed by an intravenous injection of Evans Blue dye. Ten min after injecting Evans Blue, the animal is euthanized and the dye that has extravasated into the ears is extracted overnight in formamide. The absorbance of the extracted dye is then quantified with a spectrophotometer. This method reliably results in a visual and quantifiable manifestation of a local allergic response.

Allergic responses, characterized by the activation of mast cells and basophils, are driven by the cross-linking of IgE and release of proinflammatory mediators. A quantitative assessment of allergic responses can be achieved by using Evans Blue dye to monitor changes in vascular permeability after allergen challenge.

마우스에 현지 아나필락시스 측정

Allergic responses are the result of the activation of mast cells and basophils, and the subsequent release of vasoactive and proinflammatory mediators. ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

대사 표현형을 각기 다른 미성숙, 성숙 및 Tolerogenic 수지상 세포의 생성

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

B 형 간염 바이러스 리포터 시스템 개발은 복제주기의 초기 단계를 모니터링하기

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

형광 기반의 뉴 라미니다 아제 억제 분석은 항 바이러스제의 뉴 라미니다 아제 억제제 클래스에 인플루엔자 바이러스의 감수성을 평가하기 위해

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

설치류 말라리아 기생충 무성 및 성적 혈액 단계 및 모기 단계의 현상형 분석

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

사육

요약

더 많은 비디오 탐색

이 비디오의 챕터