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.

وقد وافق كل عمل الحيوانية مكتب مختبر رعاية الحيوان في جامعة ولاية سان دييغو وأجرى بموجب المعايير البيطرية مقبولة. إناث الفئران، سن 8-16 أسابيع استخدمت، لتطوير هذا الأسلوب. 1. إعداد وداخل الصفاق حقن β استراديول <ol style=";text-align:right;direction:rtl"><li style=";te...

لتعزيز النهوض فهم التفاعلات GBS مع كل من المضيف والميكروبات الأخرى في سياق المضيف، مطلوب نموذج حيواني. يصف هذا العمل الجوانب التقنية لإنشاء GBS الاستعمار المهبل في الفئران. هذا البروتوكول يحقق&gt; 90٪ الاستعمار من الفئران من دون استخدام التخدير لتطعيم البكتيريا أو لجمع ع...

العقدية القاطعة للدر، المجموعة الثانية العقدية (GBS)، هو، البكتيريا إيجابية الجرام مغلفة التي غالبا ما تكون معزولة من الأمعاء والجهاز البولي التناسلي من البالغين الأصحاء. في 1970s، ظهرت GBS وكيلا للوفيات حديثي الولادة المعدية، مع أكثر من 7000 حالة من حالات مرض حديثي الولادة سنويا 1. المبكر بداية GBS مرض (التخلص من الذخائر المتفجرة) ويحدث في الساعات أو الأيام الأولى من الحياة، ويطرح نفسه هو الالتهاب الرئوي أو ضيق في التنفس، وغالبا ما يتطور إلى تسمم الدم، في حين أن المرض تأخر ظهور (اللد) تستتبعه بعد عدة أشهر، ويقدم مع تجرثم الدم، والتي في كثير من الأحيان التقدم إلى التهاب السحايا 2. اعتبارا من عام 2002، ومراكز السيطرة على الأمراض والوقاية منها توصي الفحص الشامل لGBS الاستعمار المهبل في أواخر الحمل والوقاية باستخدام المضادات الحيوية الولاد (IAP) لأمهات GBS الإيجابية 1. وعلى الرغم من الحد من المرض المبكر بداية إلى ما يقرب من 1000 حالة في الولايات المتحدة بسبب سنويا لIAP،يبقى GBS السبب الرئيسي للتسمم في حديثي الولادة في وقت مبكر الحدوث، وتأخر ظهور قوع يبقى بمنأى 1. سواء في الرحم، أثناء العمل، أو حتى في الحالات المتأخرة من البداية، تعرض الرضع لGBS يتطلب البقاء على قيد الحياة، مستعرضة من خلال عدد من البيئات المضيفة والحواجز، والتهرب المناعي، وفي حالة التهاب السحايا، عبور بالدم درجة عالية من التنظيم حاجز الدماغ 2. المنبع من هذه التفاعلات خبيثة داخل الوليد هو الاستعمار الأولي من القناة المهبلية الأمهات. الأم GBS معدلات الاستعمار المهبلية تتراوح 8-18٪ في البلدان المتقدمة والنامية، وبمعدل متوسط يقدر ب 12.7٪ 3،4. GBS استعمار القناة المهبلية أثناء الحمل قد يكون ثابتا، متقطعة، أو عابرة في الطبيعة بين النساء الفردية 5. ومن المثير للاهتمام، ويرتبط والأمهات العمر&gt; 36 عاما مع الاستعمار المستمرة 6. عوامل الخطر البيولوجية والاجتماعية والاقتصادية العديدة لGBS الاستعمار المهبلتم تحديدها. وتشمل العوامل البيولوجية الهضمي الاستعمار GBS وغياب الملبنة في الأمعاء. ومع ذلك، كما تم المرتبطة العرق، السمنة، والنظافة، والنشاط الجنسي مع GBS النقل المهبل 7. على الرغم من اشتهر يسبب العدوى حديثي الولادة، يسبب GBS أيضا مجموعة متنوعة من الالتهابات الأمهات على حد سواء الفترة المحيطة بالولادة وبعد الولادة. يتم زيادة GBS النقل في النساء مع تقديم المهبل 8، وفي بعض الحالات، قد يكون حتى الكيان المرض 9. بالإضافة إلى ذلك، GBS صعود الجهاز التناسلي أثناء الحمل قد يؤدي إلى عدوى داخل السلى أو شريوأمنيونيتيس 10. وعلاوة على ذلك، في ما يصل إلى 3.5٪ من حالات الحمل، GBS نشر إلى المثانة البولية أن يسبب عدوى المسالك البولية أو الجرثومية عديمة الأعراض (11). ويرتبط الجرثومية GBS خلال فترة الحمل مع زيادة خطر الاصابة بالحمى أثناء الولادة، شريوأمنيونيتيس، الولادة المبكرة، وprematurالبريد تمزق الأغشية 12. معا، ويرتبط وجود GBS داخل القناة المهبلية لالتهابات أنسجة المضيف متعددة، والقدرة على القضاء على GBS من هذا المتخصصة أمر حتمي بالنسبة للصحة الأمهات والأطفال حديثي الولادة. حتى وقت قريب، والغالبية العظمى من عمل دراسة التفاعلات GBS مع الجهاز عنقي مهبلي اقتصر على نماذج الخلايا في المختبر 13-15. وقد كشفت هذه التجارب في المختبر العوامل البكتيرية التي تعتبر مهمة للالتزام، بما في ذلك البروتينات السطحية مثل هذه الشعرة وسيرين الغنية يكرر 17،18، وكذلك النظم التنظيمية اثنين من عناصر 15،19 واستجابة النسخي العالمية للظهارة المهبل ل GBS 19. ومع ذلك، لتوضيح كامل التفاعلات المضيف ميكروب داخل القناة المهبلية، وهذا نموذج حيوان قوي أمر ضروري. أظهر العمل في وقت مبكر أن GBS يمكن استردادها من القناة المهبلية من الفئران الملقحة 20،21 والجرذان &lt;سوب&gt; 22 في كل الظروف الحوامل وغير الحوامل. في عام 2005، كان على غرار المدى القصير GBS الاستعمار المهبل في الفئران لدراسة فعالية انزيم فج التحللي لعلاج GBS المهبلية خلال فترة 24 ساعة 23. بعد عدة سنوات، وقد وضعت على المدى الطويل GBS الاستعمار المهبل نموذج الفأر لدراسة العوامل المضيفة والبكتيرية التي تحكم GBS المثابرة. وقد حدد هذا النموذج العديد من العوامل التي تسهم في GBS الاستعمار، بما في ذلك الزوائد سطح 17،18 وGBS أنظمة اثنين من عناصر 19،24. وقد ساهم هذا النموذج على تحديد آليات الاستجابة المضيف 19،25، وكان يستخدم لاختبار العديد من الاستراتيجيات العلاجية، بما في ذلك الببتيدات المناعية 26 و البروبيوتيك 27. هذا البروتوكول يعطي التوجيهات اللازمة لتطعيم GBS في القناة المهبلية الماوس وتتبع فيما بعد الاستعمار وجمع عينات لإجراء المزيد من التحليلات.

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			<td height="43" style="height:43px;width:216px;">Sesame oil&#160;</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">S3547-250ML</td>
			<td style="width:167px;"></td>
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		<tr height="46">
			<td height="46" style="height:46px;width:216px;">&#946;-Estradiol&#160;</td>
			<td style="width:91px;">Sigma Aldrich</td>
			<td style="width:119px;">E8875-1G</td>
			<td>CAUTION: Wear appropriate PPE. &#946;-estradiol can be absorbed through the skin and mucosal surfaces.&#160;</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">200 &#956;L gel loading pipette tips&#160;</td>
			<td style="width:91px;">USA Scientific</td>
			<td style="width:119px;">1252-0610</td>
			<td></td>
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			<td height="42" style="height:42px;width:216px;">Urethro-genital, sterile, calcium alginate swabs</td>
			<td style="width:91px;">Puritan</td>
			<td style="width:119px;">25-801 A 50</td>
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			<td height="42" style="height:42px;width:216px;">CHROMagar StrepB</td>
			<td style="width:91px;">DRG International</td>
			<td style="width:119px;">SB282</td>
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			<td height="42" style="height:42px;width:216px;">Todd Hewitt Broth</td>
			<td style="width:91px;">Hardy Diagnostics</td>
			<td style="width:119px;">7161C</td>
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			<td height="21" style="height:21px;width:216px;">18 G, 1.5 inch needles</td>
			<td style="width:91px;">BD</td>
			<td style="width:119px;">305199</td>
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			<td height="21" style="height:21px;width:216px;">26 G, 0.5 inch needles</td>
			<td style="width:91px;">BD</td>
			<td style="width:119px;">305111</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 mL syringes</td>
			<td style="width:91px;">BD</td>
			<td style="width:119px;">309604</td>
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			<td height="21" style="height:21px;width:216px;">1 mL syringes</td>
			<td style="width:91px;">BD</td>
			<td style="width:119px;">309659</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.45 &#956;m PVDF syringe filters</td>
			<td style="width:91px;">Whatman</td>
			<td style="width:119px;">6900-2504</td>
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			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate-Buffered Salt Solution 1X</td>
			<td style="width:91px;">Corning</td>
			<td style="width:119px;">21-031-CV</td>
			<td></td>
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a murine model of group b streptococcus vaginal colonization

العقدية القاطعة للدر (المجموعة الثانية العقدية، GBS)، هو إيجابية الجرام، المستعمر بدون أعراض في الجهاز الهضمي البشري والقناة المهبلية من 10 - 30٪ من البالغين. في الأفراد المناعة للخطر، بما في ذلك حديثي الولادة، والنساء الحوامل، وكبار السن، قد GBS التبديل إلى الممرض الغازية تسبب تعفن الدم والتهاب المفاصل والالتهاب الرئوي، والتهاب السحايا. لأن GBS هي العوامل المسببة للأمراض البكتيرية الرائدة في مجال حديثي الولادة، وتتألف الوقاية الحالية من أواخر فحص الحمل للGBS الاستعمار المهبل واللاحقة العلاج بالمضادات الحيوية الفترة المحيطة بالولادة من الأمهات GBS إيجابية. عبء المهبل GBS الثقيل هو أحد عوامل الخطر بالنسبة للأمراض الأطفال حديثي الولادة والاستعمار. للأسف، لا يعرف إلا القليل عن البلد المضيف والعوامل البكتيرية التي تشجع أو تسمح GBS الاستعمار المهبل. يصف هذا البروتوكول تقنية لإنشاء المستمر GBS الاستعمار المهبل باستخدام واحدة-β استراديول ما قبل المعالجة وأخذ العينات اليومية لتحديد مبناها البكتيريةد. ذلك مزيدا من التفاصيل أساليب لإدارة العلاجات أو الكواشف من فوائد إضافية وجمع المهبل السائل غسل وأنسجة الجهاز التناسلي. وهذا نموذج الفأر يعزز فهم التفاعل GBS المضيف ضمن بيئة المهبل، والتي سوف تؤدي إلى الأهداف العلاجية المحتملة للسيطرة الاستعمار المهبل الأمهات أثناء الحمل ومنع انتقال العدوى إلى الأطفال حديثي الولادة المعرضين للخطر. كما سيكون من مصلحة لزيادة فهمنا للتفاعلات البكتيريا في استضافة العامة في القناة المهبلية الإناث.

خلال تطوير هذا النموذج، لوحظ متعددة بشأن العوامل التي تؤثر على مدة GBS الاستعمار المهبل. لتحديد مرحلة كيف داقية في الآثار التلقيح GBS استمرار البكتيرية، ونظموا الفئران في يوم من التلقيح عن طريق السائل غسل المهبل. ويوضح الشكل 1 المراحل الأربع ?...

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

والغرض من هذا البروتوكول هو تقليد مجموعة بشرية الثانية العقدية (GBS) الاستعمار المهبل في نموذج الفئران. ويمكن استخدام هذا الأسلوب لتحقيق الاستجابة المناعية والعوامل البكتيرية المساهمة في GBS استمرار المهبلي، وكذلك لاختبار الاستراتيجيات العلاجية.

نموذج الفئران من المجموعة الثانية<em&gt; العقدية</em&gt; المهبلية الاستعمار

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.

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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.

الفئران نموذج تفعيل CD40 - B من الخلايا

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.

الاستقراء والتقييم من فئة التوحد التبديل في تنقية خلايا الفئران B

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.

تعيش خلية من التصوير<emالعصوية الرقيقة> و العقدية الرئوية الآلي باستخدام الميكروسكوب مرور الزمن

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.

بروتوكولات لتلقيح المهبل وجمع العينات في النموذج التجريبي للماوس المبيضات المهبل

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.

الكمي للاستعمار الفطرية، تولد الأبواغ، وإنتاج السموم الفطرية عن طريق اختبارات بيولوجية على النواة

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.

توصيف الاستجابات الالتهابية خلال الاستعمار داخل الشرج مع الالتهاب الرئوي العقدي

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.

التصوير غير الغازية من الاستجابة المناعية الفطرية في نموذج الزرد يرقات<em&gt; العقدية iniae</em&gt; العدوى

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.

التحديد الكمي لاستعمار الكوليرا والإسهال في النموذج الزرد الكبار

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.

تحليل القص الناجمة عن تدفق الهجرة من المنطقة الهامشية الفاري ب الخلايا في المختبر

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.

عزل واحد المجتمعات البكتيرية داخل الخلايا التي تم إنشاؤها من نموذج موريني لعدوى المسالك البولية لتحليل خلية واحدة أسفل المجرى

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