Biz mükemmel teknik yardım Elisabeth Guillemet, Christophe Buisson ve Ludovic Bridoux teşekkür etmek istiyorum. Biz ilk sistem kurulumu için Sylvie Salamitou ve Sinda Fedhila büyük ölçüde borçlu.

1. Böcek Yetiştirme Yumurtadan son evre larvalar için tüm döngü 25 ° C&#39;de yaklaşık 5 hafta sürer Bir ya da 2 hafta daha yetişkin kelebekleri elde etmek için gereklidir. <ol><li> En az 100 pupa veya yeni birleştirilen yetişkin G. yerleştirin 5 litrelik tel örgü kafes mellonella kelebekler. Erkek kelebekleri 10 ila 15 mm ölçer. Yetişkin erkek güve soluk ışık ve koyu işaretler ile bej olduğunu. 20 mm çapında Dişi kelebekler ölçüsüdür. Dişiler kahverengi / gri renk ile erkeklerden daha koyu vardır. </li><li> Yumurta-döşenmesi için kafes içinde dört katmanlı kağıt, iki paket süspanse edin. 2 gün sonra, erişkin dişi kağıtları arasındaki kenarında yumurtalarını olacaktır. </li><li> Haftada iki kez, polen ve arı balmumu içeren, hava sirkülasyonu izin ızgaraları ile yeni plastik yetiştirme kutularında yumurta kağıt paketlerini yerleştirin. Yumurtalar yaklaşık 3 gün içinde yumurtadan ve küçük larva balmumu ve polen beslenerek gelişmeye başlar. Alternatif olarak, yapay larva bir diyet consistin yerleştirilebilir500 g sıvı bal, gliserin 400 g, 100 g kurutulmuş bira mayası, 250 g buğday unu, 200 g yağsız süt tozu ve 400 g polenta bir karışımı g. Larva başına gıda uygun oranı sağlamak için, düzenli olarak yeni kutular içine larva ayırmak ve her iki günde bir yiyecek doldurmak. </li><li> Larva larva stadyum altı aşamalarında yetiştirilir. Her bir stadyum larva baş kapsül kayması ile karakterize edilir. Larvalar pupa devresi önceki son aşamaya ulaşmak zaman, onlar besleme durdurmak ve hafif ipek kozası oluşturmaya başlayabilirsiniz. </li><li> Onların koruyucu koza içinde, larva dinlendirir ve pupa haline dönüştürmek. Wax solucanlar erişkin kelebekleri ortaya bir iki hafta için pupa kalır. Yetişkin güveler içki de yemem. Çiftleşme oluşur ve balmumu solucanlar &#39;yaşam döngüsü tekrar başlar. </li><li> Patoloji testi standardize etmek için, son larva aşamasında larvaları alır. Bu aşamada onlar besleme durdurmak, yetiştirme kutusunun kapağını taşımak ve ipek üretmeye başlarlar. Bu stage 5 gün civarında sürer. </li></ol> 2. Enfeksiyon için Böcek Seçimi <ol><li> Enfeksiyonlar öncesi son dönem ağırlığı 2-3 cm uzunluğunda ve 180-250 mg olan larva, 24 saat seçin ve onları açlıktan boş bir kutu içine koymak. </li><li> Larvaları etrafında doğmakta ipek kozası çıkarın. </li></ol> 3. Bakteriyel Hazırlık <ol><li> Kob / ml 1x10 8 kob / yenmesi için ml (kob elde etmek için 10 4 koloni oluşturan birim (cfu) / ml &#39;den 10 8 kob / intra haemocoelic enjeksiyon ml ve 3x10 6 arasında değişen nihai konsantrasyonlar elde etmek için Bacillus bakteriyel süspansiyonlar hazırlayın bir LD 50) bakteri türlerine bağlıdır. Bizim durumumuzda, B. cereus &#39;un Luria-Bertani broth ortamda yetiştirilir (LB, 10 g / L tripton, 5 g / l maya ekstresi, 10 g / l NaCl) 37 ° C&#39; de karıştırma altında kadar büyüme eğrilme karşılık gelen durağan faz girmesi eğrisi. OD 600 nm (ölçünB. 1 ila 2 cereus) ve daha önceden yapılmış titrasyon eğrisi kullanarak bakteriyel konsantrasyon değerlendirmek için kullanabilirsiniz. Oda sıcaklığında ve fosfat içinde tekrar süspansiyon 10 dakika için 5000 x g&#39;de santrifüj ile hasat bakteriler (: 1M KH 2 PO 4, 1M K 2 HPO 4, 5M NaCl, pH 7.2 PBS) Tamponlu Tuzlu. Her bir larva istenen konsantrasyonda bakteriyel süspansiyon 10 ml alır. </li><li> Alternatif olarak, spor süspansiyonu enfeksiyon için hazırlanabilir. Sporlanma orta HCT (5 g / L tripton, 2 g / L kazein hidrolizat, 12.5 mM K 2 HPO 4, 12.5 mM MgSO4, 0.05 mM MnSO 4, 1.2 mM ZnSO 4, 1.2 mM Fe kültür bakterileri tarafından sporların Edinme 2 (SO4) 3,% 0.5 H2 3 gün süre ile 30 ° C &#39;de, 25 mM CaCl2) 20, 4 SO. Hasat santrifüj tarafından sporların (10,000 xg, 15 dk), ve steril distile su içinde iki kez yıkayın. Spor pel süspanseKalan vejetatif bakteriler kaldırmak için 78 az 15 dakika ° C için steril distile su ve ısı sağlar. LB agar plaklarına seri dilüsyonları kaplama ile süspansiyon numaralamak. Her bir larva istenen konsantrasyonda spor süspansiyonu 10 ml alır. </li></ol> 4. Toksin Hazırlık Cry <ol><li> B. Cry1C toksin hazırlayın thuringiensis suşu 407, geninin kodlama Cry1C taşıyan plazmid pHTF31C 21 ile dönüştürülebilir. 30 100 ml HCT ortam içinde kültür suşu ° ile 72 saat süre ile tam sporlanma C, kültür yüzey maddesinde kristal toksin üretimi ve kurtuluş sağlamak için (eritromisin 10mg/ml) antibiyotik. </li><li> 72% -79% sakkaroz degradede süpernatantı kristalleri arındırın. İlk olarak, bir 40 ml tüp içinde% 79 sakaroz 17 ml eklenir. % 79 sakkaroz üstüne% 72 sakkaroz dikkatlice tabakası 17 ml. Son olarak, katman 5-6 10X konsantre sporlu kültür ml tüp kapatın. 20,00 az 14 saat boyunca tüp Santrifüj0 xg, 4 ° C, sporlar kristaller ayırmak için. Degrade arayüzü kristalleri toplayın. Yıkamak için 25 ml soğuk steril su içinde kristal pelet yeniden süspanse edin. 20 dakika için 8000 x g&#39;de santrifüjleyin. Bu adım, kristaller yapışmasını tüm sakaroz çıkarmak için üç kez tekrarlanır. 5 ml steril su içinde yeniden süspanse edin ve kristallerin saflığı değerlendirmek için mikroskop altında dikkate alınmalıdır. </li><li> 50 mM NaOH içinde presolubilized kristal çözüm üzerinde klasik Bradford boyama ile toksin protein konsantrasyonu değerlendirin. Toksin kristaller, kullanılıncaya kadar -20 ° C&#39;de muhafaza edilebilir. </li></ol> 5. Enjektör Hazırlık <ol><li> Hassas enjeksiyon hacmi kontrol etmek için, 1 ul / sn (Şekil 1A) kurulum hızı ile otomatik şırınga pompası (örn. KD Scientific KDS 100) kullanın. </li><li> 300 ul su ile 1 ml derialtı şırınga veya 20-25 larva enfeksiyon bakteri solüsyonu (koşul başına 1 şırınga) doldurun. Dikkatle ta tarafından kabarcıklarını çıkarınşırınga pping, daha sonra şırınga ile bir 0.45 x 12 mm iğne ekleyin. </li><li> Enjektör içinde enjektör yerleştirin. </li><li> Enjekte ses seviyesini kontrol etmek boş bir tüp içinde 10 ul boş bir enjeksiyon gerçekleştirin. </li></ol> 6. Böcek Intrahaemocoelic Enjeksiyon <ol><li> Başparmak ve işaret parmağı arasında elle böcek yerleştirin. Sonra Larva deri (epiderm) (Şekil 1C) olarak enjektör sabit iğne takın. </li><li> 10 ul bakteriyel solüsyonu (spor veya vejetatif bakteriler) enjekte ederken yerde böcek tutun. </li><li> Dikkatlice iğne böcek çıkarın. </li><li> Küçük bir Petri kabı (çapı 5 cm), tabak başına 5 larvaları enfekte böcek yerleştirin. </li><li> Her bir deneysel koşul için en az 20 larva Infect. </li><li> 48 saat için bir kuluçka makinesi içinde 37 ° C &#39;de bulaşık yerleştirin. </li></ol> 7. Böcek Kuvvetleri Besleme (Yutma) <ol><li> 2 ml Eppendorf tüp, 0.2 mikrogram / &amp; m mixu; ya da spor veya vejetatif hücre süspansiyonu ile Cry1C toksin l 3x10 4 ila 10 ul başına 1x10 7 arasında değişen nihai konsantrasyonlar elde edilmiştir. </li><li> 20-25 larva enfeksiyon için bakteriyel-Cry1C toksin çözelti 300 ul, bir 30G ile 25 mm hipodermik iğne bir 1 ml&#39;lik şırınga doldurun. Şırıngadan kabarcıklarını çıkarın. </li><li> Elle yüzden onun ağzından iğne (Şekil 1D) enjektör sabit ve otomatik şırınga pompası kullanılarak bakteri solüsyonu 10 ml ile zorla beslemek girdiği böcek yerleştirin. </li><li> Her bir deneysel koşul için en az 20 larva Infect. </li><li> Küçük 5 cm Petri kabı (tabak başına 5 larva) olarak enfekte böcek yerleştirin. 48 saat için bir kuluçka makinesi içinde 37 ° C &#39;de bulaşık yerleştirin. </li></ol> 8. Kayıt ve böceklerin ölüm <ol><li> Zorla besleme veya enjeksiyon deneylerden sonra, 25 gıda olmadan Petri kabındaki larvalar tutmak ° C veya 37 ° C Larva mo Giriş48 saat süre boyunca düzenli rtality. Ölü larvalar inerttir ve genellikle siyah (Şekil 1B) çevirin. </li><li> Mortalite verileri log-probit programı 14 kullanılarak analiz edilebilir. Bu program doz ölüm eğrileri doğrusallık testleri ve öldürücü doz (LD 50) sağlar. Alternatif olarak, Prism (Grafik Pad), sağkalım analiz veya mortalite verileri gibi diğer programlar, kullanılabilir. </li></ol>

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Çıkar çatışması ilan etti.

Böceklerin ve özellikle larva olarak kullanımı, birçok patojen enfeksiyon modelleri olarak, sık hale gelmektedir. Bazı yönleri için seçim modeli yetişkin ve larva aşamasında 1,2 ikisi olarak kullanılan Drosophila (sinek modeli) olduğunu. Lepidopteran böcek G. mellonella, aynı zamanda esas olarak enjeksiyon yoluyla bakteriyel virülans test edilmesi için kullanılmıştır. Memeli patojenlere çalışılan yok edilmesi gerektiği zaman, Drosophila daha yüksek sıca...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> Wax ve polen </td><td> La Ruche Roanaise </td><td> 303000 </td><td> Herhangi bir bal üreticisi </td></tr><tr><td> Otomatik şırınga pompası </td><td> KD Scientific </td><td> KDS 100 </td><td></td></tr><tr><td> 1 ml şırınga </td><td> Terumo </td><td> BS 01T </td><td></td></tr><tr><td> İğne 0.45 x 12 mm </td><td> Terumo </td><td> NN 2613R </td><td></td></tr><tr><td> Petri 5 cm </td><td> VWR </td><td> 89000-300 </td><td></td></tr><tr><td> İğne 30G, 25 mm derialtı </td><td> Burkard Mfg Co Ltd </td><td> PDE0005 </td><td></td></tr></tbody></table>

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

Bakteriyel ölümcüllüğün çalışması genellikle uygun bir hayvan modeli gerektirir. Enfeksiyon Memeli modelleri pahalı ve etik sorunlar doğurabilir. Enfeksiyon modelleri olarak böceklerin kullanımı değerli bir alternatif sunuyor. Bu tür nematod gibi diğer non-omurgalı modeli ana karşılaştırıldığında, böcekler antimikrobiyal savunma nispeten gelişmiş sisteme sahip ve böylece daha fazla memeli enfeksiyon süreci ile ilgili bilgi üretmek olasıdır. Memeliler gibi, böcekler karmaşık bir doğuştan gelen bağışıklık sistemi 1 sahiptirler. Hemolimf hücreler mikrobik işgalciler phagocytosing veya encapsulating yeteneğine sahiptir ve hümoral yanıtlar lizozim ve küçük antibakteriyel peptidler 2,3 indüklenebilir üretimini içermektedir. Buna ek olarak, görevdeş böcek larva sindirim sistemleri ve memeli sindirim sistemi bağırsak hücrelerinin epitelyal hücreleri arasında bulunur. Son olarak, hücre adezyonu, direnç olarak bakteriyel enfeksiyon süreci için gerekli birkaç temel bileşenlerantimikrobiyal peptidler, doku bozulması ve oksidatif strese adaptasyon böcekler ve memeliler 1 hem de önemli olması muhtemeldir. Böylece, böceklerin memeli enfeksiyonlar dahil Mikrobiyal virulans faktörlerinin belirlenmesi ve tanımlanması için çok değerlikli araçlardır. Büyük bal mumu güvesi Galleria mellonella larvaları gibi Staphylococcus aureus, Proteus vulgaris gibi memeli mantar (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) ve bakteriyel patojenler içeren mikrobiyal enfeksiyonlar, geniş bir yelpazede patogenezinde dair yararlı bir içgörü sağlamak için gösterilmiştir , Serratia Pseudomonas aeruginosa, Listeria monocytogenes ve Enterococcus faecalis 4-7. Ne olursa olsun bakteri türlerinin, sonuçlar manikür yoluyla direkt enjeksiyonu ile enfekte Galleria larvaları ile tutarlı simi olanlar ile korele eldelar memeli çalışmaları: memeli modelleri zayıflatılmış olan bakteri suşları Galleria düşük virulans göstermek ve ciddi insan enfeksiyonlarına yol açan suşların Galleria modeli 8-11 oldukça kışkırtıcı. Galleria Oral enfeksiyon, belirli toksinler gibi, çok daha az kullanılan ve bileşikler ilave edilir mortalite ulaşmak için ihtiyaç vardır. G. mellonella larvaları mevcut birçok teknik avantajları: böylece bakteri tanımlanmış dozlarda enjeksiyonu sağlayan, (pupa devresi öncesi son evre larvaları yaklaşık 2 cm uzunluğunda ve ağırlığı 250 mg vardır) nispeten büyük; 20 (değişik sıcaklıklarda yetiştirilen olabilir ° 30 ° C-C) ve enfeksiyon çalışmalar, bir memeli ortam taklit deneyler sağlar, 37 ° C üstünde 12,13 ile 15 ° C arasında yürütülebilir. Buna ek olarak, böcek yetiştirme basit ve nispeten ucuz. Larvaların Enfeksiyon çeşitli yollarla, includin bakteriyel virülans izleme sağlarLD 50 14 g hesaplanması, bakteriyel sağkalım 15,16 ve enfeksiyon süreci 17 muayene ölçümü. Burada, G. tüm yaşam evrelerinde kapsayan, böceklerin yetiştirme tarif mellonella. Oral ve intra haemocoelic: Biz inokülasyon iki güzergah enfeksiyonu ayrıntılı bir protokol sağlar. Bu protokolde kullanılan bakteri örnek Bacillus cereus, mide-bağırsak yanı sıra diğer ağır lokal ya da sistemik fırsatçı enfeksiyonlar 18,19 karışmış bir Gram pozitif patojendir.

G. içine bakteri içi enjeksiyon haemocoelic mellonella birçok insan patojenleri doğuştan gelen bağışıklık faktörleri doku hasarına ve direniş ile ilgili birçok virülans faktörlerinin tanımlanması için çok yararlı olduğu kanıtlanmıştır. Bir örnek olarak, Şekil 2a, b, çeşitli doz enjeksiyonundan sonra Böcek ölümleri temsil eder cereus bakterisi (yabani tip ve mutant suşlar) 22. Şekil 2B G. enfeksiyo...

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

Büyük bal mumu güvesi larvalarının Ağız ve intra haemocolic enfeksiyonu<em&gt; Galleria mellonella</em&gt; Tarif edilmektedir. Bu böcek entomopatojenik hem de memeli fırsatçı bakteri virülans faktörü incelemek için kullanılabilir. Enfeksiyon yöntemleri ve örnekleri, böceklerin yetiştirilmesi<em&gt; In vivo</em&gt; Analiz tarif edilmektedir.

Böcek<em&gt; Galleria mellonella</em&gt; Bakteriyel Patogenez İnceleyecek Güçlü Enfeksiyon Model olarak

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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JoVE Journal

Immunology and Infection

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

31.6K Görüntüleme.  INRA, Micalis UMR1319, France. Büyük bal mumu güvesi larvalarının Ağız ve intra haemocolic enfeksiyonu Galleria mellonella Tarif edilmektedir. Bu böcek entomopatojenik hem de memeli fırsatçı bakteri virülans faktörü incelemek için kullanılabilir. Enfeksiyon yöntemleri ve örnekleri, böceklerin yetiştirilmesi In vivo Analiz tarif edilmektedir.

Video: 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-iyi Odası Slayt in vitro Biyofilm Oluşumu

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

İmmünoloji ve Enfeksiyon

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.

Farelerde Görüntü Bakteriyel Enfeksiyonlar Lusiferaz Kullanımı

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.

Bakteriyel Yapışma Çalışma Kesme Stres Tanıtımı

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 Keneler gelen tükürük, Tükürük Bezi ve hemolimf Koleksiyonu

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.

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

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.

Kullanımı<em&gt; Galleria mellonella</em&gt; Bir Model Organizma olarak Eğitim için<em&gt; Legionella pneumophila</em&gt; Enfeksiyon

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.

Bakteriyel Patojen Hücresel Enfeksiyon Soruşturma inlc salgılanmasına Görüntüleme<em&gt; Listeria monocytogenes</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.

Bir Model Olarak Zebra balığı Nitrit teratojenik potansiyelini değerlendirmek için

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.

Hamam böceği virülans, Patogenez ve uyuşturucu etkinliği araştırmak için alternatif bir Non-memeli hayvan Model tıslama Madagaskar

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 bulaşıcı hastalık virüs patogenezi okumaya bir Ex Vivo tavuk birincil Bursal hücre kültürü modeli

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