Name: a murine model of group b streptococcus vaginal colonization
Uploaded: 2016-11-16T14:00:00
Description: Watch this Scientific Journal Video about A Murine Model of Group B Streptococcus Vaginal Colonization at JoVE.com

We would like to thank the vivarium manager and staff at San Diego State University for support with animal husbandry. During this work, K.A.P. was supported by an ARCS scholarship and a fellowship from the Inamori Foundation. K.S.D. is supported by an R01 grant, NS051247, from the National Institutes of Health.

सभी जानवर काम सैन डिएगो स्टेट यूनिवर्सिटी में लैब पशु की देखभाल के कार्यालय ने मंजूरी दे दी है और स्वीकार किए जाते हैं पशु चिकित्सा मानकों के तहत आयोजित किया गया। मादा चूहों, उम्र 8 - 16 सप्ताह, इस पद्धति के विकास ?...

दोनों मेजबान के संदर्भ में मेजबान और अन्य रोगाणुओं के साथ जीबीएस बातचीत की समझ की उन्नति आगे बढ़ाने के लिए एक पशु मॉडल की आवश्यकता है। इस काम के चूहों में जीबीएस योनि उपनिवेशवाद की स्थापना के तकनीकी पह...

स्ट्रेप्टोकोकस agalactiae, समूह बी स्ट्रेप्टोकोकस (जीबीएस), एक समझाया, ग्राम पॉजिटिव जीवाणु जो अक्सर पेट और स्वस्थ वयस्कों की genitourinary पथ से अलग है। 1970 के दशक में जीबीएस संक्रामक नवजात मृत्यु दर का प्रमुख एजेंट के रूप में उभरा है, प्रतिवर्ष नवजात रोग के 7,000 से अधिक मामलों के साथ 1। पहले ही शुरू होने जीबीएस रोग (ईओडी) के पहले घंटे या जीवन के दिनों में होता है, निमोनिया या श्वसन संकट के रूप में उठता है, और अक्सर, पूति में विकसित करता है, जबकि देर शुरुआत रोग (लोद) कई महीनों के बाद ensues और bacteremia, जो अक्सर के साथ प्रस्तुत करता है 2 मैनिंजाइटिस के लिए अग्रिम। 2002 के रूप में, रोग नियंत्रण और रोकथाम के लिए केंद्र जीबीएस पॉजिटिव माताओं 1 करने के लिए देर से गर्भ और intrapartum एंटीबायोटिक प्रोफिलैक्सिस (आईएपी) में जीबीएस योनि उपनिवेश की स्थापना के लिए सार्वभौमिक स्क्रीनिंग की सिफारिश की। संयुक्त राज्य अमेरिका में लगभग 1000 मामलों में पहले ही शुरू होने रोग की कमी के सालाना आईएपी के कारण के बावजूद,जीबीएस पहले ही शुरू होने नवजात पूति का प्रमुख कारण बनी हुई है, और देर शुरुआत घटना अप्रभावित 1 रहता है। गर्भाशय में चाहे, प्रसव के दौरान, या यहां तक ​​कि देर शुरुआत मामलों में, जीबीएस के लिए नवजात जोखिम दिमागी बुखार, विनियमित अत्यधिक रक्त की क्रासिंग के मामले में मेजबान वातावरण और बाधाओं, प्रतिरक्षा चोरी के एक नंबर के माध्यम से अस्तित्व, आड़ा आवश्यकता है, और, मस्तिष्क बाधा 2। नवजात शिशु के भीतर इन विषमय बातचीत के ऊपर मातृ योनि पथ का प्रारंभिक बसाना है। मातृ जीबीएस योनि बसाना दरों 12.7% 3,4 की अनुमानित औसत दर के साथ, विकसित और विकासशील देशों में 8-18% से लेकर। गर्भावस्था के दौरान योनि मार्ग की जीबीएस उपनिवेशन, लगातार रुक-रुक कर, या अलग-अलग महिलाओं 5 के बीच में प्रकृति में क्षणिक हो सकता है। दिलचस्प है, एक मातृ उम्र&gt; 36 साल से लगातार बसाना 6 के साथ जुड़ा हुआ है। जीबीएस योनि उपनिवेश की स्थापना के लिए कई जैविक और सामाजिक-आर्थिक जोखिम कारकोंपहचान की गई है। जैविक कारकों जठरांत्र जीबीएस उपनिवेशवाद और पेट के भीतर लैक्टोबैसिलस के अभाव में शामिल हैं। हालांकि, जातीयता, मोटापा, स्वच्छता, और यौन गतिविधि भी जीबीएस योनि गाड़ी 7 के साथ संबद्ध किया गया है। हालांकि संक्रमण के कारण नवजात के लिए कुख्यात, जीबीएस भी मातृ संक्रमण के दोनों peripartum और प्रसवोत्तर की एक किस्म का कारण बनता है। जीबीएस गाड़ी योनिशोथ 8 के साथ पेश महिलाओं में बढ़ जाती है और कुछ मामलों में, यहां तक कि रोग इकाई 9 हो सकता है। इसके अतिरिक्त, गर्भावस्था के दौरान प्रजनन पथ की जीबीएस उदगम इंट्रा-एमनियोटिक संक्रमण या chorioamnionitis 10 में परिणाम हो सकता है। इसके अलावा, गर्भधारण के 3.5% तक में, जीबीएस मूत्राशय के लिए प्रसार एक मूत्र पथ के संक्रमण या स्पर्शोन्मुख बेक्टेरियूरिया 11 पैदा करने के लिए। गर्भावस्था के दौरान जीबीएस बेक्टेरियूरिया intrapartum बुखार, chorioamnionitis, अपरिपक्व प्रसव, और prematur का एक बढ़ा जोखिम के साथ जुड़ेझिल्ली 12 की ई टूटना। साथ में ले ली, योनि पथ के भीतर जीबीएस की उपस्थिति कई मेजबान ऊतकों के संक्रमण से जुड़ा हुआ है, और क्षमता इस जगह से जीबीएस समाप्त करने के लिए दोनों मातृ एवं नवजात के स्वास्थ्य के लिए आवश्यक है। अभी हाल तक, काम cervicovaginal पथ के साथ जीबीएस बातचीत की जांच के बहुमत में इन विट्रो सेल मॉडल 13-15 तक ही सीमित था। ये इन विट्रो प्रयोगों बैक्टीरियल कारक है कि पालन के लिए महत्वपूर्ण हैं, सतह प्रोटीन सहित पता चला है इस तरह के एक पिली और सेरीन अमीर दोहराता 17,18, साथ ही दो घटक नियामक प्रणाली 15,19 और योनि उपकला के वैश्विक ट्रांसक्रिप्शनल प्रतिक्रिया जीबीएस 19। हालांकि, पूरी तरह से योनि पथ के भीतर मेजबान सूक्ष्म जीव बातचीत स्पष्ट करने के लिए एक मजबूत पशु मॉडल आवश्यक है। प्रारंभिक कार्य प्रदर्शन किया है कि जीबीएस टीका चूहों 20,21 और चूहों के योनि मार्ग से बरामद किया जा सकता है &lt;sup&gt; दोनों गर्भवती और गैर गर्भवती की स्थिति में 22। 2005 में, अल्पकालिक जीबीएस योनि बसाना चूहों में मॉडलिंग की थी एक फेज अपघट्य एंजाइम की प्रभावकारिता की जांच के लिए एक 24 घंटा अवधि 23 से अधिक योनि जीबीएस के इलाज के लिए। कई साल बाद, एक लंबी अवधि जीबीएस योनि बसाना माउस मॉडल जीबीएस हठ गवर्निंग मेजबान और बैक्टीरियल कारकों का अध्ययन करने के लिए विकसित किया गया था। यह मॉडल उपनिवेशन के लिए योगदान कई कारकों जीबीएस, सतह उपांग 17,18 और जीबीएस दो घटक प्रणालियों 19,24 सहित पहचान की है। यह मॉडल मेजबान प्रतिक्रिया तंत्र 19,25 की पहचान के लिए योगदान दिया है और इम्यूनोमॉड्यूलेटरी पेप्टाइड्स 26 और 27 प्रोबायोटिक्स सहित कई चिकित्सीय रणनीतियों, परीक्षण करने के लिए इस्तेमाल किया गया था। इस प्रोटोकॉल आवश्यक मार्गदर्शन माउस योनि मार्ग में जीबीएस टीका लगाना और बाद में उपनिवेशवाद पर नज़र रखने और आगे के विश्लेषण के लिए नमूने एकत्र करने के लिए देता है।

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a murine model of group b streptococcus vaginal colonization

वयस्कों के 30% - स्ट्रेप्टोकोकस agalactiae (ग्रुप बी स्ट्रेप्टोकोकस, जीबीएस), एक ग्राम पॉजिटिव, मानव जठरांत्र संबंधी मार्ग और 10 के योनि मार्ग की स्पर्शोन्मुख उपनिवेशवादी है। प्रतिरक्षा-समझौता व्यक्तियों, नवजात शिशुओं सहित, गर्भवती महिलाओं, और बुजुर्गों में, जीबीएस के कारण पूति, गठिया, निमोनिया, और दिमागी बुखार एक आक्रामक रोगज़नक़ के लिए स्विच कर सकते हैं। क्योंकि जीबीएस नवजात शिशुओं के एक अग्रणी जीवाणु रोगज़नक़ है, वर्तमान प्रोफिलैक्सिस जीबीएस योनि उपनिवेशवाद और जीबीएस पॉजिटिव माताओं के बाद peripartum एंटीबायोटिक उपचार के लिए देर से गर्भ स्क्रीनिंग के शामिल है। भारी जीबीएस योनि बोझ दोनों नवजात रोग और उपनिवेशवाद के लिए एक जोखिम कारक है। दुर्भाग्य से, थोड़ा मेजबान और बैक्टीरियल कारक है कि को बढ़ावा देने या जीबीएस योनि उपनिवेश की स्थापना की अनुमति के बारे में जाना जाता है। इस प्रोटोकॉल एक भी β-एस्ट्राडियोल पूर्व उपचार और दैनिक नमूने का उपयोग बैक्टीरियल लोआ निर्धारित करने के लिए लगातार जीबीएस योनि उपनिवेशवाद की स्थापना के लिए एक तकनीक का वर्णनघ। यह अधिक जानकारी के तरीकों अतिरिक्त उपचारों या ब्याज की अभिकर्मकों प्रशासन के लिए और योनि पानी से धोना तरल पदार्थ और प्रजनन पथ ऊतकों एकत्र करने के लिए। इस माउस मॉडल योनि वातावरण में जीबीएस मेजबान बातचीत, जो गर्भावस्था के दौरान मातृ योनि उपनिवेशवाद को नियंत्रित करने और कमजोर नवजात शिशु को संचरण को रोकने के लिए संभावित चिकित्सीय लक्ष्यों को बढ़ावा मिलेगा की समझ को आगे होगा। यह भी महिला योनि मार्ग में सामान्य जीवाणु मेजबान बातचीत के बारे में हमारी समझ को बढ़ाने के लिए ब्याज की हो जाएगा।

इस मॉडल के विकास के दौरान, कई टिप्पणियों कारक है कि जीबीएस योनि उपनिवेशवाद की अवधि को प्रभावित के बारे में किए गए थे। टीका प्रभावों जीबीएस बैक्टीरियल हठ पर कैसे estrous चरण का निर्धारण करने के ल...

Watch this Scientific Journal Video about A Murine Model of Group B Streptococcus Vaginal Colonization at JoVE.com

A Murine Model of Group B Streptococcus Vaginal Colonization

इस प्रोटोकॉल के उद्देश्य से एक murine मॉडल में मानव समूह बी स्ट्रेप्टोकोकस (जीबीएस) योनि उपनिवेशवाद की नकल करने के लिए है। इस विधि मेजबान प्रतिरक्षा प्रतिक्रिया और जीबीएस योनि हठ के लिए योगदान दे बैक्टीरियल कारकों की जांच करने के लिए, साथ ही चिकित्सीय रणनीतियों का परीक्षण करने के लिए इस्तेमाल किया जा सकता है।

ग्रुप &#39;बी&#39; के एक murine मॉडल<em&gt; स्ट्रेप्टोकोकस</em&gt; योनि औपनिवेशीकरण

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

The overall goal of this animal model is to establish group B Streptococcus, or GBS colonization of the murine vaginal tract. This method can help us understand key questions about group B Streptococcal colonization, such as, what are the bacterial factors that are important for colonization, as well as how host responses impact vaginal persistence. The main advantage of this technique is that inoculation, typically, results in greater than 90 percent colonization without the use of anesthesia, immunosuppressants, or thickening agents.Begin by vortexing 0.5 milligrams of beta-Estradiol, per mouse, in a 15 milliliter conical tube until all of the lumps are removed and the beta-Estradiol appears as a fine powder. Next, syringe filter 100 microliters of sesame oil, per mouse, into the tube through a 0.45 micron strainer, and vortex the tube again. When the hormone is distributed as a homogenous suspension throughout the oil, draw the reagent into a new, 10 milliliter syringe and attach an 18 gauge, one inch needle.Aliquot 100 microliters of the suspension into one, one milliliter tuberculin syringe, per mouse, and equip each tuberculin syringe with a sterile, 26 gauge, half inch needle. Then, deliver 100 microliters of beta-Estradiol into the lower left or right quadrant of the peritoneal cavity of each animal. On the same day as the injection, Grow a five milliliter, overnight liquid culture of the GBS strain of interest in Todd Hewitt broth, or THB, at 37 degrees Celsius.The next morning, subculture the GBS at a one to 10 volume in fresh THB for a two to three hour incubation at 37 degrees Celsius, until the bacteria reach mid-log phase. Then, transfer the the subculture into a sterile, 15 milliliter conical tube, and centrifuge the bacterial cells. Resuspend the pellet in 200 microliters of sterile PBS.Then, bring the total volume of the suspension up to one milliliter of PBS per 10 mice, to reach an OD600 of 0.4 in a new, five milliliter culture tube. Transfer the culture to a new, 15 milliliter tube, and re-pellet the bacteria. Then, resuspend the cells in PBS at one tenth the original volume.Reserve 50 microliters of the bacteria for serial dilution and plating on THB agar, to determine the exact CFU of the inoculum. Then, draw 10 microliters of GBS into a 200 microliter gel-loading pipette tip, and manually secure the loose skin at the scruff of the neck, of the first animal, between the thumb and index finger, followed by immobilization of the tail. Insert the pipette tip 5 to 10 millimeters into the vaginal lumen, and dispense the entire volume of the inoculum.Then, immediately release the scruff and lift the tail to elevate the hind end of the animal, while walking the front paws on a hard surface. After a minute, visually inspect the vaginal opening for any inoculum backflow. Just prior to swabbing, pre-wet the cotton in PBS, and restrain one of the mice as just demonstrated.Insert the swab 10 millimeters into the vaginal lumen, and gently rotate four times clockwise, and four times counterclockwise, while applying slight pressure to the vaginal wall. Place the swab into a 1.5 milliliter microcentrifuge tube containing 100 microliters of PBS, and vortex the tube and swab for about 15 seconds to release the bacteria from the cotton fibers. Then, serially dilute each sample in PBS, and plate 20 microliters of one to 10 through one to 10, 000 dilutions on differential medium agar plate, prepared per the manufacturer's instructions, for incubation at 37 degrees Celsius for 24 hours.GBS colonies will appear as either bright pink or mauve in color. To collect the vaginal lavage, use a 200 microliter gel-loading pipette tip to dispense 20 microliters of PBS into the vaginal lumen. Gently pipette the entire volume up and down four times within the lumen.Then, using the same tip, withdraw the entire volume. For downstream lavage fluid analysis, dispense the lavage into a 0.7 milliliter microcentrifuge tube. For tissue dissection homogenization, spray down the ventral abdomen of a euthanized animal with 70 percent ethanol, and use sterile scissors to open the abdominopelvic cavity.Displace the intestines so that the reproductive tract is exposed. Then, cut both uterine horns, mid-length, between the uterine body and ovaries. Using scissors and forceps, separate the visceral fat, membranes, and urinary bladder from the reproductive tract, moving the tissues caudally.Then, transversely cut the vagina as close to the vulva as possible, to separate the reproductive tract from the body. Transfer the in-tact reproductive tract into a sterile Petri dish, and use a new razor blade to separate the uterus from the cervix in a single, transverse cut. Separate the cervix from the vagina with another transverse cut.Then, use sterile forceps to transfer each of the tissues into individual, pre-weighed, two milliliter tubes containing 500 microliters of PBS and homogenizing beads. Weigh each 2 milliliter tube to determine the tissue weights. Then, tightly seal the screw cap tubes and homogenize the tissues, for one minute, at the maximum speed, in a tissue homogenizer.To quantify the bacterial load, serially dilute 25 microliters of tissue homogenate and plate one to 10 through one to 10, 000 dilutions on differential medium agar plates, for a 24 hour incubation, at 37 degrees Celsius. To store samples for citokine quantification, freeze the homogenates at minus 20 degrees Celsius. In these images, the four stages of the mouse estrus cycle, as determined by wet-mount vaginal lavage fluid, are demonstrated.Mice inoculated at the proestrus stage were colonized with GBS longer than any other stage of estrus, particularly those in diestrus at the time of inoculation. Sustained estrus promotes GBS A909 persistence in CD-1 mice with a 90 percent colonization observed two weeks post inoculation. In a typical experiment with one dose of beta-Estradiol prior to inoculation, only 40 to 50 percent of the mice were colonized one week post inoculation, with a recovered mean GBS that mimics the percent of colonization.Demonstrating that maintaining continuous estrus promotes GBS vaginal persistence in the majority of CD-1 mice. GBS A909 persists in the vaginal tract in outbred CD-1 mice and inbred FVB mice for, approximately, one week. Whereas the majority of inbred BALB/c and C57BL/6 mice become colonized at one week for a month or longer.Visualization of GFP positive GBS in inoculated animals demonstrates the presence of GBS adhered to the murine vaginal epithelium and in close proximity to other native vaginal flora. It is important to prevent backflow, during inoculation, to ensure that all mice become colonized. Once mastered, this technique can be completed in one to two hours, depending on the number of animals.This model allows for researchers in the field of host/pathogen interactions, to explore the ability of bacteria, like group B strep, to colonize the female reproductive tract. This procedure can be used to test novel compounds or probiotic treatments to answer additional questions about therapeutic interventions.

a-murine-model-of-group-b-streptococcus-vaginal-colonization

Research

JoVE Journal

Immunology and Infection

A Murine Model of Group B Streptococcus Vaginal Colonization

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

बी कोशिकाओं की CD40 सक्रियण के murine मॉडल

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the immunoglobulin locus undergoes a series of genetic modifications to alter the binding capacity and effector function of secreted antibodies. This process is highlighted by a genomic recombination event known as class switch recombination (CSR) in which the default IgM antibody isotype is substituted for one of IgG, IgA, or IgE. Each isotype possesses distinct effector functions thereby making CSR crucial to the maintenance of immunity. Diversification of the immunoglobulin locus is mediated by the enzyme activation-induced cytidine deaminase (AID). A schematic video describing this process in detail is available online (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). AID's activity and the CSR pathway are commonly studied in the assessment of B cell function and humoral immunity in mice. The protocol outlined in this report presents a method of B cell isolation from murine spleens and subsequent stimulation with bacterial lipopolysaccharide (LPS) to induce class switching to IgG3 (for other antibody isotypes see Table 1). In addition, the fluorescent cell staining dye Carboxyfluorescein succinimidyl ester (CFSE) is used to monitor cell division of stimulated cells, a process crucial to isotype switching 1, 2. The regulation of AID and the mechanism by which CSR occurs are still unclear and thus in vitro class switch assays provide a reliable method for testing these processes in various mouse models. These assays have been previously used in the context of gene deficiency using knockout mice 3. Furthermore, in vitro switching of B cells can be preceded by viral transduction to modulate gene expression by RNA knockdown or transgene expression 4-6. The data from these types of experiments have impacted our understanding of AID activity, resolution of the CSR reaction, and antibody-mediated immunity in the mouse.

Following antigen exposure, subpopulations of activated B cells undergo a process known as class switch recombination (CSR) to produce antibody isotypes with distinct effector functions. The protocol outlined in this report explains how CSR can be induced and analyzed in vitro for the purposes of studying B cell function.

प्रेरण और murine बी कक्ष शुद्ध में कक्षा स्विच पुनर्संयोजन के मूल्यांकन

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

लाइव सेल की इमेजिंग बेसिलस subtilis और स्ट्रैपटोकोकस निमोनिया स्वचालित माइक्रोस्कोपी समय चूक का उपयोग

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

योनि और प्रायोगिक माउस मॉडल में नमूना संग्रह टीका के लिए प्रोटोकॉल Candida योनिशोथ

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

फफूंद की औपनिवेशीकरण, रेणूजनक और mycotoxins का उत्पादन की मात्रा कर्नेल bioassays का उपयोग

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

स्ट्रेप्टोकोकस निमोनिया के साथ इंट्रानासाल उपनिवेशीकरण के दौरान भड़काऊ प्रतिक्रियाओं का लक्षण वर्णन

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

की एक zebrafish लारवल मॉडल में सहज प्रतिरक्षा प्रतिक्रिया के गैर इनवेसिव इमेजिंग<em&gt; स्ट्रेप्टोकोकस iniae</em&gt; संक्रमण

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the aquatic environment, where it interacts with a variety of higher aquatic species. Vertebrate fish are known to be an environmental host and are a potential V. cholerae reservoir in nature. Both V. cholerae and the teleost fish species Danio rerio, commonly known as zebrafish, originate from the Indian subcontinent, suggesting a long-standing interaction in aquatic environments. Zebrafish are an ideal model organism for studying many aspects of biology, including infectious diseases. Zebrafish can be easily and rapidly colonized by V. cholerae after exposure in water. Intestinal colonization by V. cholerae leads to the production of diarrhea and the excretion of replicated V. cholerae. These excreted bacteria can then go on to colonize new fish hosts. Here, we demonstrate how to assess V. cholerae-intestinal colonization in zebrafish and how to quantify V. cholerae-induced zebrafish diarrhea. The colonization model should be useful to researchers who are studying whether genes of interest may be important for host colonization and/or for environmental survival. The quantification of zebrafish diarrhea should be useful to researchers studying any intestinal pathogen who are interested in exploring zebrafish as a model system.

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Zebrafish are a natural Vibrio cholerae host and can be used to recapitulate and study the entire infectious cycle from colonization to transmission. Here, we demonstrate how to assess V. cholerae colonization levels and quantify diarrhea in zebrafish.

बढ़ाता Vibrio हैजा औपनिवेशीकरण और दस्त वयस्क Zebrafish मॉडल में

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Murine सीमांत क्षेत्र बी कोशिकाओं के कतरनी प्रवाह प्रेरित प्रवासन का विश्लेषण इन विट्रो में

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected in the urinary tract. The protocol can be broadly divided into three sections: the infection, bladder epithelial cell harvesting, and mouth micropipetting to isolate individual infected epithelial cells. The isolated epithelial cell contains viable bacterial cells and is nearly free of contaminating extracellular bacteria, making it ideal for downstream single-cell analysis. The time taken from the start of infection to obtaining a single intracellular bacterial community is about 8 h. This protocol is inexpensive to deploy and uses widely available materials, and we anticipate that it can also be utilized in other infection models to isolate single infected cells from cell mixtures even if those infected cells are rare. However, due to a potential risk in mouth micropipetting, this procedure is not recommended for highly infectious agents.

This protocol describes a simple method of isolating single, infected-bladder epithelial cells from a murine model of urinary tract infection.

एकल-कोशिकीय जीवाणु समुदायों का अलगाव जो अनुप्रवाह एकल कोशिका विश्लेषण के लिए मूत्र पथ के संक्रमण के म्यूरिन मॉडल से उत्पन्न होता है ।

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...