우리는 우수한 기술 지원을 위해 엘리자베스 Guillemet, 크리스토프 Buisson와 Ludovic Bridoux 감사드립니다. 우리는 초기 시스템 설정을위한 Sylvie Salamitou 및 Sinda Fedhila 께 무한한 빚을 수 있습니다.

1. 곤충 양육 계란의 마지막 instar의 애벌레에 대한 모든 과정이 25 ° C.에 약 5 주 정도 1 개 또는 2 개 추가 주는 성인 나비를 얻기 위해 필요합니다. <ol><li> 최소 100 pupae 또는 새로 합병 성인 G.를 배치 5 리터 와이어 메쉬 케이지의 mellonella 나비. 남자 나비는 10~15mm을 측정합니다. 성인 남성이 나방은 희미한 빛과 어두운 표시가있는 베이지 색입니다. 20mm 주변의 여성 나비 측정. 암컷은 갈색 / 회색과 수컷보다 어두운입니다. </li><li> 계란 부설에 대한 우리에 네 계층 종이 두 팩을 일시 중지합니다. 이일 후, 성인 여성은 신문 사이의 가장자리에 알을 낳는 것입니다. </li><li> 일주일에 두 번, 꽃가루와 벌 왁스가 포함 된 공기가 순환하게하기 위해 격자로 새로운 플라스틱 양육 상자에 계란 종이 팩을 넣습니다. 알은 약 3 일 부화 작은 애벌레는 왁스와 화분에 공급하여 개발 시작합니다. 또한, 애벌레는 인공 다이어트 consistin에 배치 할 수 있습니다500g 액체 꿀, 400g의 글리세린, 100g 건조 맥주 효모, 250g의 밀가루, 200g 탈지 분유와 400g 폴렌타 죽의 혼합의 g. 유충 당 음식의 적절한 비율을 제공하기 위해 정기적으로 새로운 상자에 애벌레를 분리 모든 이틀 음식을 보충. </li><li> 애벌레는 유충 경기장의 여섯 단계에서 키운 있습니다. 각 경기장은 유충의 머리 캡슐의 미끄럼이 특징입니다. 애벌레는 pupation 전에 마지막 단계에 도달하면, 그들은 먹이를 중지하고 가벼운 실크 누에 고치를 구축 시작합니다. </li><li> 자신의 보호 고치에서 애벌레 휴식하고 pupae로 변환 할 수 있습니다. 왁스 웜은 성인 나방에 등장하는 1~2주의 번데기 단계에 남아 있습니다. 성인 나방은 술도 먹지 않습니다. 짝짓기가 발생하고 왁스 웜 &#39;수명주기가 다시 시작됩니다. </li><li> 병리 검정을 표준화하기 위해 마지막 애벌레의 단계에서 유충을. 이 단계에서 사람들이 먹이를 중지, 양육 상자의 덮개에 이동 실크를 생산하기 시작합니다. 이 Stage 5 일 주위 지속됩니다. </li></ol> 2. 감염에 대한 곤충 선택 <ol><li> 감염 전에 마지막으로 instar 무게에 2-3cm 길이 180-250 밀리그램 아르 애벌레,, 24 시간을 선택하고을 굶어 빈 상자에 넣어 두 었어요. </li><li> 애벌레 주변의 초기 실크 누에 고치를 제거합니다. </li></ol> 3. 세균 준비 <ol><li> CFU / ML 1x10 8 CFU / 섭취에 대한 ML을 (CFU는 구하는 방법 10 4 식민지 성형 단위 (CFU) / ML에서 10 8 CFU / 내부 haemocoelic 분사에 대한 ML과 3x10 6에서까지 최종 농도를 얻기 위해 세균 세균 현탁액을 준비 LD 50) 박테리아 종에 따라 달라집니다. 우리의 경우, B.에 세레 우스는 Luria-Bertani 국물 매체에서 재배됩니다 (LB, 10g / L tryptone, 5g / L 효모 추출물, 10g / L NaCl) 37의 ° C 교반 이하까지 성장의 안으로 굽음에 해당하는 고정 단계로 진입, 곡선. OD 600 nm의를 (측정B. 1 사이 2 세레 우스)과 이전에 한 적정 곡선을 사용하여 세균 농도를 평가하는 데 사용합니다. 객실 온도와 인산의 resuspend 10 분 5,000 XG에서 원심 분리하여 수확 박테리아 (: 1M KH 2 PO 4, 100 K 2 HPO 4, 5M NaCl, pH를 7.2 PBS) 식염 버퍼링. 각 유충 원하는 농도에서 세균 현탁액의 10 μl를 받게 될 것입니다. </li><li> 또한, 포자 현탁액은 감염을 준비 할 수 있습니다. sporulation 매체 HCT (5 G / L tryptone, 2 G / L 카세인 가수 분해물, 12.5 MM K 2 HPO 4, 12.5 MM MgSO 4, 0.05 MM MnSO 4, 1.2 MM ZnSO 4, 1.2 MM 철의 배양 세균을하여 포자를 얻을 2 (SO 4) 3, 0.5 % H 2 3 일 30 ° C 25 MM CaCl 2) 20, 4 SO. 수확 원심 분리하여 포자 (10,000 XG, 15 분) 및 멸균 증류수에 두 번 씻는다. 포자 펠를 Resuspend남은 식물 세균을 제거하는 78시 15 분 ° C에 멸균 증류수와 열에 표시 할 수 있습니다. LB 한천 플레이트에 시리얼 dilutions을 도금하여 정지 수계. 각 유충 원하는 농도에 포자 현탁액의 10 μl를 받게 될 것입니다. </li></ol> 4. 독소 준비 눈물 <ol><li> B.에서 Cry1C 독소를 준비 thuringiensis 변형 407은 유전자 인코딩 Cry1C를 들고 플라스미드 pHTF31C 21 변환. 30 100 ML의 HCT 매체의 문화 변형 °와 72 시간을위한 C 전체 sporulation, 문화 표면에 뜨는에 독소 크리스탈 생산과 해방을 허용하도록 (에리스로 마이신 10mg/ml) 항생제. </li><li> 72% -79 %의 자당 기울기에 표면에 뜨는에서 결정을 정화. 먼저 40 ML 튜브에 79 % 자당 17 ML를 추가합니다. 79 % 자당 위에 72%의 자당의주의 층 17 ML. 마지막으로, 레이어 5-6 10X 집중 sporulated 문화의 ML하고 관을 닫습니다. 20,00에서 14 시간 동안 튜브를 원심 분리기0 XG, 4 ° C는 포자의 결정을 분리합니다. 기울기 인터페이스에서 결정을 수집합니다. 그것을 씻어 25 ML 차가운 멸균 물에 크리스탈 펠렛을 Resuspend. 20 분에 8,000 XG에 원심 분리기. 이 단계는 결정에 집어 넣는 모든 자당을 제거하려면 세 번 반복됩니다. 5 ML 멸균 물에 결정을 Resuspend하고 순결을 평가하기 위해 현미경으로 관찰한다. </li><li> 50 MM NaOH에 presolubilized 크리스탈 솔루션에 고전 브래드 포드 착색에 의해 독소 단백질 농도를 평가합니다. 독소 결정은 사용 할 때까지 -20 ° C에 저장할 수 있습니다. </li></ol> 5. 주입기 준비 <ol><li> 정확한 주입 볼륨을 제어하려면, 1 μl / 초 (그림 1A)에 설치 속도로 자동 주사기 펌프 (예 : KD 과학 KDS 100)을 사용합니다. </li><li> 300 μl 물 1 ML의 피하 주사기 또는 20-25 애벌레의 감염 세균 솔루션 (조건 당 1 주사기)를 입력합니다. 조심스럽게 타하여 거품을 제거주사기 pping 다음 주사기에 0.45 X 12mm 바늘을 첨부합니다. </li><li> 주입기에 주사기를 삽입합니다. </li><li> 주입 된 볼륨을 제어 할 빈 튜브에 10 μl의 빈 주사를 수행합니다. </li></ol> 6. 곤충 Intrahaemocoelic 사출 <ol><li> 엄지와 집게 손가락 사이에 수동으로 곤충를 놓습니다. 그런 다음 유충 피부 (표피) (그림 1C)에 분사기에서 해결 된 바늘을 삽입합니다. </li><li> 10 μl 세균 솔루션을 (포자 또는 식물 박테리아) 주입하면서 장소에서 곤충세요. </li><li> 조심스럽게 바늘에서 곤충을 제거합니다. </li><li> 작은 페트리 접시 (직경 5cm), 음식 당 5 유충에 감염된 곤충를 놓습니다. </li><li> 각 실험 조건에 대한 최소한 20 애벌레를 감염. </li><li> 48 시간에 대한 보육에서 37 ° C에서 요리를 놓으십시오. </li></ol> 7. 곤충 강제 수유 (섭취) <ol><li> 2 ML Eppendorf 튜브에 0.2 μg / &amp; m를 섞어U; 중 포자 나 식물 세포 현탁액과 Cry1C 독소 내가 3x10 4에서 10 μl 당 1x10 7에 이르기까지 최종 농도를 얻을 수 있습니다. </li><li> 20-25 애벌레의 감염 세균 - Cry1C 독소 솔루션 300 μl과 30g의 25 밀리미터의 피하 주사기 바늘을 1 ML의 주사기를 채우십시오. 주사기에서 거품을 제거합니다. </li><li> 수동 그래서 그 입을 바늘 (그림 1D) 분사기에서 해결하고, 자동화 된 주사기 펌프를 사용하여 세균 솔루션의 10 μl로 강제 먹이를 입력하는 곤충를 놓습니다. </li><li> 각 실험 조건에 대한 최소한 20 애벌레를 감염. </li><li> 작은 5cm 페트리 접시 (접시 당 5 유충)에 감염된 곤충를 놓습니다. 48 시간에 대한 보육에서 37 ° C에서 요리를 놓으십시오. </li></ol> 8. 녹화 곤충 사망률 <ol><li> 힘 수유 또는 주입 실험을 한 후, 25에서 음식없이 배양 접시에있는 애벌레를 유지 ° C 또는 37 ° C. 애벌레의 몰리브덴을 확인48 시간 기간 동안 정기적으로 rtality. 죽은 유충은 불활성이며 일반적으로 검은 색을 (그림 1B) 설정합니다. </li><li> 사망률 데이터는 로그 Probit 프로그램 14을 사용하여 분석 할 수 있습니다. 이 프로그램은 선량 사망률 곡선의 선형성을 시험하고 치명적인 복용 (LD 50)을 제공합니다. 또한, 이러한 프리즘 (그래프 패드), 생존을 분석하거나 사망률 데이터 같은 다른 프로그램이 사용할 수 있습니다. </li></ol>

<ol>
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O., Ribier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generalized+transduction+in+Bacillus+thuringiensis+var.+berliner+1715,+using+bacteriophage+CP54.">Generalized transduction in Bacillus thuringiensis var. berliner 1715, using bacteriophage CP54.</a> Ber. J. Gen. Microbiol. 121, 203-212 (1980).</li><li>Sanchis, V., Agaisse, H., Chaufaux, J., Lereclus, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+new+insecticidal+Bacillus+thuringiensis+recombinant+strains+by+using+the+sporulation+non-dependent+expression+system+of+cryIIIA+and+a+site+specific+recombination+vector.">Construction of new insecticidal Bacillus thuringiensis recombinant strains by using the sporulation non-dependent expression system of cryIIIA and a site specific recombination vector.</a> J. Biotechnol. 48, 81-96 (1996).</li><li>Tran, S. L., Guillemet, E., Gohar, M., Lereclus, D., Ramarao, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CwpFM+(EntFM)+is+a+Bacillus+cereus+potential+cell+wall+peptidase+implicated+in+adhesion,+biofilm+formation+and+virulence.">CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation and virulence.</a> J. Bacteriol. 192, 2638-2642 (2010).</li><li>Tran, S. L., 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=Hemolysin+II+is+a+Bacillus+cereus+virulence+factor+that+induces+apoptosis+of+macrophages.">Hemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages.</a> Cell Microbiol. 13, 92-108 (2011).</li><li>Fedhila, S., 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=Comparative+analysis+of+the+virulence+of+invertebrate+and+mammalian+pathogenic+bacteria+in+the+oral+insect+infection+model+Galleria+mellonella.">Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella.</a> J. Invertebr. Pathol. 103, 24-29 (2010).</li><li>Daou, N., 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=IlsA,+a+unique+surface+protein+of+Bacillus+cereus+required+for+iron+acquisition+from+heme,+hemoglobin+and+ferritin.">IlsA, a unique surface protein of Bacillus cereus required for iron acquisition from heme, hemoglobin and ferritin.</a> PLoS Pathog. 5, e1000675 (2009).</li><li>Mason, K. L., 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=From+commensal+to+pathogen:+translocation+of+Enterococcus+faecalis+from+the+midgut+to+the+hemocoel+of+Manduca+sexta.">From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta.</a> MBio. 2, e00065-00011 (2011).</li><li>Goldsmith, M. R., Shimada, T., Abe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetics+and+genomics+of+the+silkworm,+Bombyx+mori.">The genetics and genomics of the silkworm, Bombyx mori.</a> Annu. Rev. Entomol. 50, 71-100 (2005).</li><li>Fraser, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insect+transgenesis:+current+applications+and+future+prospects.">Insect transgenesis: current applications and future prospects.</a> Annu. Rev. Entomol. 57, 267-289 (2012).</li></ol>

관심 없음 충돌이 선언 없습니다.

곤충, 특히 애벌레의 단계의 사용은 여러 병원체에 대한 감염 모델로, 잦은되고 있습니다. 일부 측면에 대한 선택의 모델은 성인과 애벌레의 무대 1,2 모두로 사용 Drosophila (플라이 모델)입니다. lepidopteran 곤충 G. mellonella는 주로 주입하여 세균 독성을 시금에 사용되었습니다. 포유류의 병원체가 공부 할 때 Drosophila보다 높은 온도를 (37 ° C 이상) (최대 25 ° C) 참아의 장점...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> 왁스와 화분 </td><td> 라 루슈 Roanaise </td><td> 303000 </td><td> 모든 벌꿀 생산 </td></tr><tr><td> 자동 주사기 펌프 </td><td> KD 과학 </td><td> KDS 100 </td><td></td></tr><tr><td> 주사 한 ML </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> 니들 0.45 X 12mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> 페트리 접시 5cm </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> 바늘 30g의, 25 밀리미터 피하 </td><td> Burkard 제조 (주) </td><td> PDE0005 </td><td></td></tr></tbody></table>

the insect galleria mellonella as a powerful infection model to investigate bacterial pathogenesis

박테리아 독성의 연구는 종종 적절한 동물 모델이 필요합니다. 감염의 포유류의 모델은 비용이 많이 들고 있으며, 윤리적 문제를 제기 할 수 있습니다. 감염 모델과 같은 곤충의 사용은 가치있는 대안을 제공합니다. 같은 nematodes와 같은 다른 비 척추 동물 모델 호스트에 비해 곤충은 항균 방어 비교적 고급 시스템을 가지고 있으며 따라서 더 포유류의 감염 과정에 관련 정보를 생성 할 가능성이 있습니다. 포유 동물과 마찬가지로, 곤충은 복잡한 타고난 면역 시스템 1을 보유하고 있습니다. hemolymph에있는 세포 미생물 침략자를 phagocytosing하거나 캡슐화 할 수 있으며, 체액 반응은 라이소자임 작은 항균 펩티드 2,3의 inducible 생산이 포함되어 있습니다. 또한, 유추는 곤충 애벌레의 midguts와 포유류의 소화 시스템의 장 세포의 상피 세포 사이에 발견된다. 마지막으로, 이러한 세포 부착, 저항 등의 세균 감염 과정에 필수적인 몇 가지 기본 구성 요소항균 펩티드, 조직 저하 및 산화 스트레스에 대한 적응은 곤충과 포유류 한 모두에 중요 할 가능성이 있습니다. 따라서, 곤충 포유류의 감염에 관련된 미생물 독성 요소의 식별 및 특성에 대한 여러 균을 혼합 한 도구입니다. 큰 왁스 나방 갤러리아 mellonella의 애벌레는 이러한 포도상 구균, 프로테우스의 vulgaris와 같은 포유류의 곰팡이 (Fusarium oxysporum, 누룩 곰팡이 fumigatus, 칸디다 알비 칸스) 및 세균성 병원체를 포함한 미생물 감염, 다양한 범위의 pathogenesis에 유용한 통찰력을 제공하기 위해 표시되었습니다 , Serratia marcescens 모나스 aeruginosa, 리스테리아 monocytogenes 또는 Enterococcus faecalis 4-7. 상관없이 박테리아 종, 결과는 큐티클을 통해 직접 분사에 감염 갤러리아 애벌레와 함께 지속적으로 시미의 그것과 상관 관계를 획득고맙다 포유류 연구 : 포유류의 모델 감쇠 아르 박테리아 변종은 갤러리아에서 낮은 독성을 보여줍니다, 그리고 심각한 인간 감염을 일으키는 변종도 갤러리아 모델 8-11에 매우 유독합니다. 갤러리아의 구강 감염이, 특정 독소와 같은 훨씬 덜 사용 및 화합물 추가됩니다는 사망률에 도달 할 필요합니다. G. mellonella의 애벌레 존재하는 몇 가지 기술적 인 장점 : 사람들이 따라서 박테리아의 정의 복용의 주입을 사용 (pupation 전에 마지막 instar의 애벌레는 약 2 cm 길이, 무게 250 밀리그램입니다) 비교적 크기, 그들은 20 일 (다양한 온도에서 키운 할 수 있습니다 ° 30 ° C로 C) 및 감염 연구는 포유류의 환경을 모방 실험을 허용, 37 ° C 12,13 위를 15 ° C 사이에 실시 할 수 있습니다. 또한, 곤충 양육은 쉽고 상대적으로 저렴합니다. 애벌레의 감염은 여러 수단, includin에 의해 박테리아 독성을 모니터링 할 수 있습니다LD 50 14 g 계산, 박테리아 생존 15,16 및 감염 과정 17 시험의 측정. 여기, 우리는 G.의 모든 삶의 단계를 덮고, 곤충의 양육을 설명 mellonella. 구두 및 내부 haemocoelic : 우리는 접종의 두 경로로 감염에 대한 자세한 프로토콜을 제공합니다. 이 프로토콜에 사용되는 세균 모델은 바실루스 세레 우스, 위장뿐만 아니라 다른 심각한 지역 또는 조직 기회 감염 18,19에 연루 그램 긍정적 인 병원체이다.

G.에 박테리아의 내부 haemocoelic 주입 mellonella는 여러 사람이 병원균의 타고난 면역 요인에 조직 손상 및 저항을 다루는 많은 독성 요소의 식별을 위해 매우 유용한 입증되었습니다. 예를 들어, 그림 2A는 B.의 다양한 복용의 주입 후 곤충 사망률을 나타냅니다 세레 우스 균 (야생 유형과 돌연변이 변종) 22. 그림 2B는 G 감염 후 세균 ?...

Watch this Scientific Journal Video about The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis at JoVE.com

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

큰 왁스 나방의 애벌레의 구강 내부 haemocolic 감염<em&gt; 갤러리아 mellonella</em&gt;이 설명되어 있습니다. 이 벌레는 entomopathogenic뿐만 아니라 포유류의 기회 박테리아의 독성 요인을 연구하는 데 사용할 수 있습니다. 감염 방법과의 예는 곤충의 양육<em&gt; 생체 내</em&gt; 분석에 설명되어 있습니다.

곤충<em&gt; 갤러리아 mellonella</em&gt; 세균 Pathogenesis를 조사 할 수있는 강력한 감염 모델로

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

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Immunology and Infection

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

31.6K 조회수.  INRA, Micalis UMR1319, France. 큰 왁스 나방의 애벌레의 구강 내부 haemocolic 감염 갤러리아 mellonella이 설명되어 있습니다. 이 벌레는 entomopathogenic뿐만 아니라 포유류의 기회 박테리아의 독성 요인을 연구하는 데 사용할 수 있습니다. 감염 방법과의 예는 곤충의 양육 생체 내 분석에 설명되어 있습니다.

비디오: The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

In vitro Biofilm Formation in an 8-well Chamber Slide

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. Biofilms are community-associated bacteria attached to a surface and encased in a matrix. The role of the extracellular matrix is multifaceted, including facilitating nutrient acquisition, and offers significant protection against environmental stresses (e.g. host immune responses). In an effort to acquire a better understanding as to how the bacteria within a biofilm respond to environmental stresses we have used a protocol wherein we visualize bacterial biofilms which have formed in an 8-well chamber slide. The biofilms were stained with the BacLight Live/Dead stain and examined using a confocal microscope to characterize the relative biofilm size, and structure under varying incubation conditions. Z-stack images were collected via confocal microscopy and analyzed by COMSTAT. This protocol can be used to help elucidate the mechanism and kinetics by which biofilms form, as well as identify components that are important to biofilm structure and stability.

In vitro Biofilm Formation in an 8-well Chamber Slide

This article describes the procedure for the formation and visualization of a bacterial biofilm grown within an 8-well chamber slide

8 잘 회의소 슬라이드에서 체외의 Biofilm 형성에

The chronic nature of many diseases is attributed to the formation of bacterial biofilms which are recalcitrant to traditional antibiotic therapy. ...

연구

면역학 및 감염병학

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

세균성 접착 연구에 전단 응력을 소개합니다

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

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 ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS로 인한 세균 경쟁을 모니터링 할 비주얼 분석

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae&#39;s immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

The larva of the wax moth Galleria mellonella was recently established as an in vivo model to study Legionella pneumophila infection. Here, we demonstrate fundamental techniques to characterize the pathogenesis of Legionella in the larvae, including inoculation, measurement of bacterial virulence and replication as well as extraction and analysis of infected hemocytes.

사용<em&gt; 갤러리아 mellonella</em&gt; 모델 생물로 공부하기<em&gt; 레지오넬라 뉴모 필라</em&gt; 감염

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Bacterial intracellular pathogens can be conceived as molecular tools to dissect cellular signaling cascades due to their capacity to exquisitely manipulate and subvert cell functions which are required for the infection of host target tissues. Among these bacterial pathogens, Listeria monocytogenes is a Gram positive microorganism that has been used as a paradigm for intracellular parasitism in the characterization of cellular immune responses, and which has played instrumental roles in the discovery of molecular pathways controlling cytoskeletal and membrane trafficking dynamics. In this article, we describe a robust microscopical assay for the detection of late cellular infection stages of L. monocytogenes based on the fluorescent labeling of InlC, a secreted bacterial protein which accumulates in the cytoplasm of infected cells; this assay can be coupled to automated high-throughput small interfering RNA screens in order to characterize cellular signaling pathways involved in the up- or down-regulation of infection.

Imaging InlC Secretion to Investigate Cellular Infection by the Bacterial Pathogen Listeria monocytogenes

Listeria monocytogenes is a Gram positive bacterial pathogen frequently used as a major model for the study of intracellular parasitism. Imaging late L. monocytogenes infection stages within the context of small-interfering RNA screens allows for the global study of cellular pathways required for bacterial infection of target host cells.

세균성 병원체에 의해 세포 감염을 조사하기 위해 InlC 분비 이미징<em&gt; 리스테리아</em

Bacterial intracellular  pathogens can be conceived as molecular tools to dissect cellular signaling  cascades due to their capacity to exquisitely ...

Zebrafish as a Model to Assess the Teratogenic Potential of Nitrite

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of nitrate to nitrite by gut and salivary bacteria. However, in other mammalian studies, high nitrite levels do not cause birth defects, although they can lead to poor reproductive outcomes. Thus, the teratogenic potential of nitrite is not clear. It would be useful to have a vertebrate model system to easily assess teratogenic effects of nitrite or any other chemical of interest. Here, we demonstrate the utility of zebrafish (Danio rerio) to screen compounds for toxicity and embryonic defects. Zebrafish embryos are fertilized externally and have rapid development, making them a good model for teratogenic studies. We show that increasing the time of exposure to nitrite negatively affects survival. Increasing the concentration of nitrite also adversely affects survival, whereas nitrate does not. For embryos that survive nitrite exposure, various defects can occur, including pericardial and yolk sac&#160;edema, swim bladder noninflation, and craniofacial malformation. Our results indicate that the zebrafish is a convenient system for studying the teratogenic potential of nitrite. This approach can easily be adapted to test other chemicals for their effects on early vertebrate development.

Exposure to teratogens may cause birth defects. Zebrafish are useful for determining the teratogenic potential of chemicals. We demonstrate the utility of zebrafish by exposing embryos to various levels of nitrite and also at different times of exposure. We show that nitrite can be toxic and cause severe developmental defects.

High nitrate levels in the environment may&#160;result in congenital defects or miscarriages in humans. Presumably, this is due to the conversion of ...

The Madagascar Hissing Cockroach as an Alternative Non-mammalian Animal Model to Investigate Virulence, Pathogenesis, and Drug Efficacy

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can be utilized as an alternative animal model for the study of virulence, host-pathogen interaction, innate immune response, and drug efficacy. Details for the rearing, care and breeding of the hissing cockroach are provided. We also illustrate how it can be infected with bacteria such as the intracellular pathogens Burkholderia mallei, B. pseudomallei, and B. thailandensis. Use of the hissing cockroach is inexpensive and overcomes regulatory issues dealing with the use of mammals in research. In addition, results found using the hissing cockroach model are reproducible and similar to those obtained using mammalian models. Thus, the Madagascar hissing cockroach represents an attractive surrogate host that should be explored when conducting animal studies.

We present a protocol to utilize the Madagascar hissing cockroach as an alternative non-mammalian animal model to conduct bacterial virulence, pathogenesis, drug toxicity, drug efficacy, and innate immune response studies.

마다 가스 카 르 독성, 병 인, 및 약물 효능을 조사 하기 위해 대체 비 포유류 동물 모델로 서 치찰음의 바퀴벌레

Many aspects of innate immunity are conserved between mammals and insects. An insect, the Madagascar hissing cockroach from the genus Gromphadorhina, can ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Bursal 전염병 바이러스 병 인을 공부 하는 Ex Vivo 닭 기본 Bursal 세포 배양 모델

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...