Yazarlar Avrupa Komisyonu Yedinci Çerçeve Programı (PIOF-GA-2009-255466) ve İrlanda Sağlık Araştırma Kurulu&#39;nun (HRA_POR/2010/138) Bu yazının ilgili destek kabul etmek istiyoruz. Lux etiketli E. coli Dr Cormac Gahan, University College Cork bir tür hediye oldu.

1. Tümör İndüksiyon <ol><li> Rutin tümör indüksiyon için, serum içermeyen kültür ortamı 200 ul içinde süspansiye edilmiş hücreler asgari tümörojenik doz serbest enfeksiyonun yan 6-8 haftalık dişi Balb / C veya atimik fareler içine MF1-nu/nu (sc) deri altından enjekte edilmiştir n 21-gauge enjektör iğnesi kullanarak = 6 (Harlan, Oxfordshire, UK) (1 x 10 6 4T1 hücreleri). Bir haemocytometer ve tripan mavi boyasıyla çıkarma (Gibco) kullanılarak görsel sayımı ile belirlenir olarak aşılama için kullanılan hücre canlılığı% 95&#39;ten daha fazla idi. </li><li> Tümör kurulmasının ardından, tümörleri büyümeye ve gelişmeye izin verildi ve iki haftalık takip edildi. Tümör hacmi tümörün en uzun çapı ve b çapı için en uzun çapı dik olan ve formül V = (ab 2) Π / 6, göre hesaplanmıştır. </li></ol> 2. Bakteriyel Hazırlık <ol><li> Bu protoco kullanılan bakteri suşul E. oldu coli K-12 MG1655, non-protein-toksin salgılayan bakterileri tarafından tespit edilmesini sağlar bir luxABCDE kodlama plazmid barındıran, zorlanma BLI. E. Entegre luxABCDE içeren coli MG1655, 37 aerobik olarak yetiştirilen ° C LB ortamı (Sigma-Aldrich, İrlanda) 300 ug / ml eritromisin (Em) ile desteklenmiştir. MG1655 ve bioluminescent türevi plazmid p16S lux bünye P YARDIM luxABCDE operon 1 içeren kullanılarak oluşturuldu. </li><li> Fareler için uygulama için hazırlanması için, kültürler orta log fazı (600 nm&#39;de optik yoğunluk) 200 rpm&#39;de bir çalkalama kabında 37 ° C&#39;de LB ortamı içinde inkübe edildi. Bakteriler, (6000 x g 5 dakika) santrifüj ile toplanır, PBS (Sigma) ile yıkanmış ve PBS içinde seyreltilmiş ve 1 x 10 7 koloni oluşturan birim (CFU) / intravenöz için mL, ya da gavaj için 1 x 10 10. </li></ol> 3. Bakteriyel Dairesition <ol><li> Tümör hacmi yaklaşık 100 mm 3 ulaştığı zaman fareler rasgele deney grubuna ayrıldı. Intravenöz için, ölçülü fareler her bir 28G şırınga iğnesi kullanarak lateral kuyruk ven içine doğrudan enjekte 100 ul 10 6 hücre aldı. Her bir inokulum canlı sayımı geriye dönük kaplama ile tespit edilmiştir. </li><li> GYTE kolonizasyon çalışmaları için 10 9 bakteri hücrelerinin oral üç gün üst üste, gavaj yoluyla fare başına 100 ul uygulanmıştır. Önceden mevcut komensal bakteri seviyeleri önce ağızdan sonda 1 başlamasından 7 gün için fare içme suyu içinde 5 mg / ml streptomisin ilavesiyle besleme önce düşürülmüştür. </li></ol> 4. Biyoparlaklık Görüntüleme <ol><li> In vivo BLI görüntüleme 2B IVIS100 (Kaliper) kullanılarak yapıldı. Tanımlanmış zaman noktalarında sonrası bakteriyel yönetimi olarak, farelerin Kaliper en XGI-8 Gaz Anestezi kullanarak uyutulduktan% 3 Isofluorane, ve tüm-vücut görüntü analiz sistemi ile yüksek duyarlılık 2-5 dakika boyunca sistem 100 IVIS uygulandı. </li><li> 3D görüntüleme için, anestezi fareler dorsal görüntüleme için optik görüntüleme sistemi (IVIS Spektrum, kaliper) içinde bir fare görüntüleme servis yerleştirildi. 3D optik rekonstrüksiyon için bakteriyel lusiferaz sinyalinin görüntü elde etmek için, 500-580 nm arasında değişen emisyon filtresi dalga gürültü oranı sinyali maksimize etmek için filtre başına 3-4 dk bin 16 edinimi kez kullanılmıştır. Bu görüntü elde etme sırası bir parçası olarak, yapılandırılmış bir ışık görüntü yükseklik haritası tanımlamak için elde edilmiştir. Bu harita giriş diffüz ışık görüntüleme tomografi (DLIT) negatif olmayan en küçük kareler optimizasyon 2 kullanarak bir 3D optik görüntü oluşturmak için kullanılan rekonstrüksiyon algoritmaları oldu. </li><li> Görüntü Analizi: ilgi bölgeler belirlenmeli ve Yaşam Image yazılımı (Kaliper) kullanılarak ölçüldü. </li></ol> 5. TemsilciSonuçlar Bu çalışmada, patojenik olmayan ortakçı bakteri E.coli K-12 MG1655 luxABCDE operon ifade IV sc 4T1 ksenogreft tümörleri taşıyan farelere uygulandığında oldu. Bakteriyel lux sinyalinin farelerde tümörlerin özellikle saptandı sonrası IV-yönetim (Şekil 2). Örnek fareler elde edilen bakteri kültürü iyileşme uygun bakteri sayısı ve tespit ışık miktarı (Şekil 3) arasında doğrusal bir ilişki varlığını doğrular. GIT içinde oral olarak uygulandığında, bakterilere ve in vivo olarak da 3D BLI kullanılarak elde edilir. <img src="/files/ftp_upload/4318/4318fig1.jpg" alt="Şekil 1" fo:content-width="4in" fo:src="/files/ftp_upload/4318/4318fig1highres.jpg"/> Şekil 1. Protokol Timeline. Subkutan tümörler farelerde indüklenen ve bakteriler tümör gelişimi (100 mm 3) üzerine uygulanır. Canlı fareler BLI im vardırçeşitli zaman noktalarında sonrası bakteriyel idaresi (oklar tipik kez gösterilecek) de yaş. <img src="/files/ftp_upload/4318/4318fig2.jpg" alt="Şekil 2," /> Şekil 2. E. İdaresi Tümör taşıyan fareler için coli MG1655 luxABCDE. Subkutan 4T1 tümörlerin MF1 nu / nu farelerine ve E&#39;de indüklenen edildi coli MG1655 luxABCDE tümör gelişimi üzerine uygulanmıştır. Her hayvan lateral kuyruk ven içine doğrudan enjekte 10 6 hücre aldı. Fare örnek kurban farelerde tümör canlı bakteri sonraki kurtarma (cfu) ile çalışma sırasında dört zaman noktalarında (siyah noktalar z ekseni ve görüntüler) (çubuk grafik) de görüntülendi. Bakteriyel numaraları ve özellikle tümörlerde zaman (temsilcisi fare zaman noktası başına resimli) üzerinde saptandı plazmid gen ekspresyonu artış. <a href="http://www.jove.com/files/ftp_upload/4318/4318fig2large.jpg" target="_blank">büyük bir rakam görmek için buraya tıklayın</a> .<img src="/files/ftp_upload/4318/4318fig3.jpg" alt="Şekil 3" /> Şekil 3,. İntratümöral Bakteriyel Numaraları Ve Biyoparlaklık Arasındaki İlişki. Canlı bakteri tümörleri çeşitli zaman noktalarında sonrası intravenöz az BLI izleyen tümörlerden ex vivo bakteriyel kültür tarafından numaralandırılan. In vivo bioluminesce birimleri göre bakteri sayı değerleri (cfu), oturum grafikle edilir. Bakteri sayımı ve bakteriyel biyolüminesans sinyalleri arasında sağlam bir ilişki gözlenmektedir R 2 = 0,9717 1. <img src="/files/ftp_upload/4318/4318fig4.jpg" alt="Şekil 4," /> Şekil 4. Mürin gastrointestinal 3D IVIS Görüntü E. tarafından kolonileştirildiğini coli MG1655. farelerin GYTE E. 10 9 cfu oral tarafından kolonize edildi üç gün üst üste coli. Kolonize fare 3D tomografi alınan bir numuneden izole görüntü gösterilir.3 boyutlu görüntüler anatomik kayıt sağlanmasında iskeletin bir dijital fare atlası göstermektedir. E. coli MG1655 biyolüminesans düşük yeşil görünür, ve yüksek düzeyde mor.

<ol>
	<li>Cronin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+in+vivo+bioluminescent+imaging+for+the+study+of+bacterial+tumour+targeting.">High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting.</a> PLoS One. 7, e30940 (2012).</li><li>Kuo, C., Coquoz, O., Troy, T. L., Xu, H., Rice, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+in+vivo+bioluminescent+sources+based+on+multispectral+imaging.">Three-dimensional reconstruction of in vivo bioluminescent sources based on multispectral imaging.</a> J. Biomed. Opt. 12, 024007 (2007).</li><li>Tangney, M., Ahmad, S., Collins, S. A., O'Sullivan, G. 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Çıkar çatışması ilan etti.

Gen terapi bağlamında, hasta için terapötik gen ulaştırılması için biyolojik ajanların kullanımı büyük söz 3-5 göstermiştir. Gibi virüsler, bakteriler ve doğal biyolojik özelliklerini, özellikle kanser bağlamında, hücreler ya da dokulara etkili DNA aktarma izin verir. Bakterilerin doğal olarak yeteneğine sahip olduğu gösterilmiştir için sistemik olarak ya da lokal olarak replikasyon dış, yüksek düzeyde (invazif olmayan türler) ya da tümör hücreleri (patojen) içinde idare...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> 4T1 hücre çizgisi </td><td> ATCC </td><td> CRL-2539 </td><td> Kendiliğinden ortaya çıkan alınan BALB / c meme tümörü türetilen sinjenik meme kanseri modelinde </td></tr><tr><td> DMEM </td><td> Sigma-Aldrich </td><td> D6429 </td><td> Dulbecco Modifiye Kartal Orta </td></tr><tr><td> PBS </td><td> Sigma-Aldrich </td><td> D8537 </td><td> Fosfat Tamponlu Salin </td></tr><tr><td> Xenogen IVIS </td><td> Kaliper Yaşam Bilimleri </td><td></td><td> 2D görüntüleme için IVIS 100; 3D için IVIS Spektrum. </td></tr><tr><td> Luria Broth Miller (LB) </td><td> Sigma-Aldrich </td><td> L2542 </td><td> E. Büyüme orta coli </td></tr&gt; <tr><td> Eritromisin </td><td> Sigma-Aldrich </td><td> E5389 </td><td> Antibiyotik </td></tr><tr><td> Streptomisin </td><td> Sigma-Aldrich </td><td> S9137 </td><td> Antibiyotik </td></tr><tr><td> MF1nu/nu fareler </td><td> Harlan (UK) </td><td> 069 (nu) / 070 (nu / +) </td><td> HSD: atimik Nude-Foxn1nu </td></tr><tr><td> Balb / c fareleri </td><td> Harlan (UK) </td><td> 066 </td><td> Haplotipi: H-2 d </td></tr><tr><td> Gavaj iğne </td><td> Vet-tech Solutions (UK) </td><td> DE009 </td><td> 22G x 38mm düz gavaj iğnesi </td></tr><tr><td> IV enjeksiyon için Şırınga </td><td> BD BioSciences&#39;ı </td><td> 309309-1 mL </td><td> 28 G x ½ inç mikro-ince IV iğne ile insülin şırınga. </td></tr><tr><td> Tümör inokülasyon için Şırınga </td><td> Braun </td><td> 9161376V </td><td> Omnifix 26 G x &amp;frac12; inç iğne </td></tr></tbody></table>

bioluminescent bacterial imaging in vivo

Bu video kanseri için gen ve hücre tedavisi bakterilerin kullanımı ile ilgili bir vurgu ile, canlı farelerde bakteriyel ticareti çalışma için tüm vücut bioluminesce görüntüleme (BLI) kullanımı anlatılmaktadır. Bakteriler sistemik uygulamayı takiben tümörler içinde tercihen büyümek için doğal bir yeteneği sahip, kanser tedavisi için vektör çekici bir sınıf mevcut. Bakteriler bakteri ve aynı zamanda tümörün sitelerin lux gen kasetinin izni BLI algılama ifade için tasarlanmıştır. Zaman içinde tümörler içinde yer ve bakteri düzeylerini kolayca kontrol edilebilir, iki ya da üç boyutlu olarak gözlemlenmiştir. Yöntem bakteri türleri ve tümör ksenogreft türleri geniş bir aralığı için geçerlidir. Bu makale, subkutan tümör taşıyan fareler içinde biyolüminesans bakteri analizi için protokol açıklanmaktadır. BLI ile gastrointestinal sistem (GIT) içinde bakterilere görselleştirilmesi de tarif edilmektedir. Bu güçlü ve ucuz, gerçek zamanlı görüntüleme stratejisi temsilcisits belirli kanser, özellikle gen tedavisi araştırma, ve enfeksiyöz hastalık bağlamında in vivo bakteri çalışma için ideal bir yöntem. Bu video lux-etiketli E. incelemek için prosedür ana hatlarıyla IVIS sistemi ile mekansal ve zamansal okuma başarılabilir kullanan BLI gösteren canlı farelerde coli.

Watch this Scientific Journal Video about Bioluminescent Bacterial Imaging In Vivo at JoVE.com

Bioluminescent Bacterial Imaging In Vivo

Bu makale idaresi anlatılmaktadır<em&gt; Lux-etiketlendi</em&gt; Fareler bakteri ve sonraki<em&gt; In vivo</emIVIS biyolüminesans görüntüleme kullanılarak&gt; analizi.

Bioluminescent Bakteriyel Görüntüleme<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Research

JoVE Journal

Immunology and Infection

Bioluminescent Bacterial Imaging In Vivo

15.1K Görüntüleme.  University College Cork. Bu makale idaresi anlatılmaktadır Lux-etiketlendi Fareler bakteri ve sonraki In vivo analizi.

Video: Bioluminescent Bacterial Imaging In Vivo

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

Canlı bir Host Transgenik Leishmania Parazitlerinin in vivo Imaging

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

İmmünoloji ve Enfeksiyon

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Lökosit Homing Göç ve non-invaziv Görüntüleme In vivo</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Tüm Yaprak Görüntüleme kullanarak Bakteriyel Hücreler Live Stoma Tepki Değerlendirilmesi

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Yılları arasında sinerjistik Araştırma Bioluminescent Görüntüleme kullanma Streptococcus pneumoniae Bebek Fare ve İnfluenza A Virüsü

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Kategori Algılama Bir Bakteriyel Patojenler &#39;Bioluminescent&#39; Muhabir Faj

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

Bir Nöro-İnvazif encephalitic Virüs Vivo Görüntüleme Sistemleri (IVIS) Algılama

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

In vivo Görüntüleme Yöntemi Akut ve Kronik İltihap ayırt

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Üretim ve Çok fenotipik Yüksek içerik Eleme<em&gt; Coxiella burnetii</em&gt; Transposon Mutantlar

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In Vitro ve İn Vivo Modeli Gemi Duvar Altında Akış Koşulları Bakteriyel Yapışma Eğitim için

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...