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.

All animal work was approved by the Office of Lab Animal Care at San Diego State University and conducted under accepted veterinary standards. Female mice, age 8 - 16 weeks, were used for the development of this method.
1. Preparation and Intraperitoneal Injection of &#946;-estradiol
<ol>
	<li>Measure out &#946;-estradiol (0.5 mg/mouse) on weigh paper while wearing appropriate personal protective equipment (PPE). CAUTION: &#946;-estradiol can be absorbed through the skin and mucosal surfaces.</li>
	<li>Transfer &#946;-estradiol to a 15-ml conical tube and vortex until all lumps are removed and the &#946;-estradiol is a fine powder. Draw up sesame oil (100 &#181;l/mouse) into a 10-ml syringe. Syringe-filter sesame oil into the 15-ml conical tube containing the &#946;-estradiol using a 0.45-&#181;m filter. Vortex the 15-ml conical tube until the &#946;-estradiol is a homogenous suspension in the sesame oil.</li>
	<li>Draw up the &#946;-estradiol suspension into a new 10-ml syringe. With an 18 G, 1-in. needle, aliquot 100 &#181;l of the suspension into 1-ml tuberculin syringes. Prepare one syringe for each mouse. Place a new, sterile 26 G, &#189;-in. needle on each tuberculin syringe.</li>
	<li>Administer 0.5 mg of the &#946;-estradiol suspended in 100 &#181;l of sesame oil (5 mg/ml) to each mouse 24 hr prior to bacterial inoculation. Inject each mouse within the peritoneal cavity, in the lower abdominal quadrants, just to the right or left of the midline, as previously described28. 
	NOTE: No cleaning or clipping of the injection site is necessary.</li>
</ol>
2. Vaginal Inoculation with GBS
<ol>
	<li>One day prior to inoculation, grow a 5-ml overnight liquid culture of a GBS strain of interest, such as A909 (serotype Ia), in Todd Hewitt broth (THB) at 37 &#176;C.</li>
	<li>Subculture the GBS overnight culture at a 1:10 volume into fresh THB and incubate at 37 &#176;C. Grow the bacteria to mid-log phase (OD600 = 0.4-0.5). NOTE: This will typically take 2 - 3 hr, depending on the strain of GBS.</li>
	<li>Transfer the subculture to a sterile 15-ml conical tube and pellet bacteria at 3,000 &#215; g for 5 min. Aspirate the supernatant. Resuspend the bacterial pellet in 200 &#181;l of sterile phosphate-buffered saline (PBS).</li>
	<li>Using the resuspended pellet, bring 3 - 5 ml of PBS (1 ml per 10 mice) to exactly OD600 = 0.4 in a new 5-ml culture tube. This will be a concentration of ~ 1 &#215; 108 colony forming units (CFU)/ml. Transfer to a new 15-ml conical tube and re-pellet the bacteria at 3,000 &#215; g for 5 min. Aspirate the supernatant.</li>
	<li>Resuspend the pellet in PBS at 1/10 the original volume. For example, if 3 ml of OD600 = 0.4 was pelleted, then resuspend it in 300 &#181;l of PBS. NOTE: This is the final bacterial suspension (~ 1 &#215; 109 CFU/ml) used for animal inoculation.</li>
	<li>Reserve 50 &#181;l of this suspension for serial dilution and plating on THB agar to determine the exact inoculum.</li>
	<li>Inoculate each mouse with 10 &#181;l of the final bacterial suspension so that 1 &#215; 107 CFU is administered to each mouse.
	<ol>
		<li>To inoculate, restrain the mouse manually by securing the loose skin at the scruff of the neck between the handler&#39;s thumb and index finger and then immobilizing the tail, as described previously28.</li>
		<li>Draw up 10 &#181;l of the GBS prepared in step 2.6 into a 200-&#181;l gel loading pipette tip. Insert the tip 5 to 10 mm into the vaginal lumen and dispense the 10 &#181;l of inoculum. 
		NOTE: Gel loading tips are preferred over standard 200-&#181;l tips to minimize the risk of organ trauma or injury, particularly in younger or smaller mice.</li>
		<li>Immediately following inoculation, release the scruff of the neck and elevate the hind end of the mouse, lifting the mouse by the tail and walking the front paws on a hard surface for ~ 1 min.</li>
		<li>Visually inspect the vaginal opening for any backflow of inoculum. If backflow is observed, a fresh pipette tip may be used to manipulate or enlarge the vaginal opening, facilitating uptake of the backflow into the lumen. Additionally, backflow may be aspirated via pipette and re-inoculated. 
		NOTE: If administering topical agents, probiotic organisms, or proteins of interest, a volume up to 20 &#181;l in a physiologic buffer may be given in the vaginal tract.</li>
	</ol>
	</li>
</ol>
3. Swabbing the Vaginal Lumen to Quantify GBS Load
<ol>
	<li>Prepare one 1.5-ml micro-centrifuge tube per mouse by adding 100 &#181;l of PBS. Just prior to swabbing, pre-wet the swab in PBS.</li>
	<li>Restrain the mouse as described in step 2.7.1 and insert the swab 10 mm into the vaginal lumen. Gently rotate the swab 4 times clockwise and 4 times counter clockwise, applying slight pressure to the vaginal wall.</li>
	<li>Transfer the swab to the 1.5-ml microcentrifuge tube with 100 &#181;l of PBS. Prior to plating, vortex the microcentrifuge tube for ~15 sec to release the bacteria from the swab. Serially dilute each sample in PBS and plate 20 &#181;l of dilutions 1:10 through 1:10,000 on differential medium agar plates prepared per the manufacturer&#39;s instructions. Incubate the plates at 37 &#176;C for 24 hr. GBS colonies will appear either bright pink or mauve in color. Other endogenous flora will be inhibited, or will appear as blue, white, or grey colonies.</li>
</ol>
4. Collecting Vaginal Lavage Fluid
<ol>
	<li>Restrain the mouse as described in step 2.7.1. Using a 200-&#181;l gel loading pipette tip, pipet 20 &#181;l of PBS into the vaginal lumen. Gently pipet the entire volume up and down 4 times within the lumen, and then withdraw the entire volume in the same pipette tip. NOTE: If the lavage fluid is thick with mucous, a standard 200 &#181;l pipette tip can be used to collect the final lavage fluid.</li>
	<li>If saving lavage fluid for cytokine analysis or CFU quantification, dispense into a 0.7-ml microcentrifuge tube. If determining the stage of estrus, dispense at least 5 &#181;l of lavage fluid onto a glass slide and observe the cells under 100X magnification on a light microscope. NOTE: For examples of estrous stages, see Figure 1.</li>
</ol>
5. Tissue Dissection and Homogenization
<ol>
	<li>For each mouse, prepare three 2-ml screw cap tubes (one for each tissue: vagina, cervix, and uterus). Fill each tube with ample (0.4 - 0.5 g) 1.0 mm zirconia beads to cover the conical-shaped bottom of the tube.</li>
	<li>Autoclave the prepared tubes prior to collecting tissue for 30 min at 121 &#176;C, especially if quantifying bacterial load. Add 500 &#181;l of sterile PBS to each tube. Weigh each tube after autoclaving and record for future reference to calculate recovered tissue weight.</li>
	<li>Sacrifice the mouse using approved institutional methods such as CO2 asphyxiation and cervical dislocation. Spray down the ventral abdomen with 70% EtOH. With sterile scissors, open the abdominopelvic cavity, lift the back skin and abdominal muscles, and displace the intestines so that the reproductive tract is exposed.</li>
	<li>Sterilize the scissors with 70% EtOH, wipe clean if necessary, and cut both uterine horns mid-length between the uterine body and ovaries. Using scissors and forceps, separate visceral fat, membranes, and the urinary bladder away from the reproductive tract, moving caudally.</li>
	<li>Sterilize scissors as described in step 5.4. Transversely cut the vagina as close to the vulva as possible to separate the reproductive tract from the body. Lift and remove the intact reproductive tract and place it in a sterile Petri dish.</li>
	<li>Using a new razor blade, separate the uterus from the cervix in one transverse cut. Sterilize the razor blade with 70% EtOH, and then separate the cervix from the vagina in one transverse cut. NOTE: There will be a minimal amount of uterine tissue still attached to the cervix. There will be a minimal amount of vaginal tissue still attached to the cervix.</li>
	<li>Using sterile forceps, transfer each of the tissues into their respective 2-ml tubes containing PBS and homogenizing beads. Clean the forceps with 70% EtOH in between handling each tissue.</li>
	<li>Weigh each 2-ml screw cap tube and subtract the original weight of tube to determine the tissue weight. Tissue weights typically vary between 20 - 100 mg. Tightly seal the screw cap tubes and homogenize the tissues for 1 min at maximum speed in a tissue homogenizer.</li>
	<li>To quantify bacterial load, serially dilute 25 &#181;l of tissue homogenate and plate dilutions 1:10 through 1:10,000 on differential medium agar plates. Incubate the plates as described in step 3.3. To store samples for cytokine quantification, freeze tissue homogenates at -20 &#176;C.</li>
</ol>

The authors have nothing to disclose.

To further the advancement of the understanding of GBS interactions with the both the host and other microbes within the context of the host, an animal model is required. This work describes the technical aspects of establishing GBS vaginal colonization in mice. This protocol achieves &#62; 90% colonization of mice without the use of anesthetics to inoculate bacteria or to collect swab samples, immune-suppressants to enable colonization, vaginal pre-washing, or additives to thicken the inoculum. Moreover, this model demo...

ストレプトコッカス・アガラクティエ 、B群連鎖球菌 （GBS）は、頻繁に腸と健康な成人の尿生殖路から隔離されているカプセル化された、グラム陽性細菌です。 1970年代には、GBSは毎年、新生児の病気の7,000以上の例1で、感染新生児死亡率の主要な薬剤として登場しました。早期発症GBS疾患（EOD）が肺炎や呼吸困難として生じる、最初の数時間または生命の日に発生し、多くの場合、遅発性疾患（LOD）のに対し、敗血症に発展する数ヶ月後に続いて起こると頻繁に菌血症、と提示します2髄膜炎に進みます。 2002年のように、疾病管理予防センター（CDC）は、GBS陽性の母親1に妊娠後期や分娩に抗生物質予防法（IAP）でGBS膣植民地化のための普遍的なスクリーニングを推奨しています。毎年IAPによる米国では約千例に早期発症疾患の減少にもかかわらず、GBSは、早発型新生児敗血症の主要な原因のままであり、遅発性の発生は1影響を受けないままです。子宮内かどうかは、労働者の間に、あるいは遅発性の場合には、GBSの新生児の暴露は髄膜炎、高度に規制血液の交差した場合に、ホスト環境や障壁、免疫回避の数を介して生存、横断を必要とし、脳関門2。新生児内のこれらの病原性の相互作用の上流には、母体の膣管の初期の植民地です。母体のGBS膣コロニー形成率は12.7％3,4の推定平均速度で、先進国と途上国での8から18パーセントの範囲です。妊娠中の膣管のGBSコロニー形成は、個々の女性の5の間で自然の中で、一定の断続的な、または一過性であってもよいです。興味深いことに、母体の年齢&gt; 36年には、永続的植民地化6に関連しています。 GBS膣植民地化のための多数の生物学的および社会経済的なリスク要因同定されています。生物学的因子は、胃腸GBSのコロニー形成および腸内で乳酸桿菌の不在が含まれます。しかし、民族性、肥満、衛生、および性的活動もGBS膣キャリッジ7に関連しています。 新生児の感染を引き起こすための悪名高いが、GBSはまた、母体の感染症の両方の周産期および産後の様々な原因となります。 GBSキャリッジも疾患実体9とすることができる、いくつかのケースでは、膣炎8を呈する女性で増加しています。また、妊娠中の生殖器官のGBSの昇天は、羊膜内感染症または絨毛羊膜炎10になることがあります。また、妊娠の3.5％までに、GBSは、尿路感染症や無症候性細菌尿11を引き起こすために膀胱に発信しています。妊娠中のGBS細菌は、分娩の発熱、絨毛羊膜炎、早産、およびprematurのリスク増加と関連しています膜12の電子破裂。まとめると、膣管内のGBSの存在は、複数の宿主組織の感染症にリンクされており、このニッチからGBSを排除する能力の両方の妊産婦と新生児の健康のために不可欠です。 最近まで、頸膣管とGBSの相互作用を調べる作業の大半は、in vitro細胞モデル13-15に制限されていました。これらのin vitroの実験では、表面タンパク質を含む接着のために重要である細菌の要因を、明らかにしたような線毛およびセリンリッチリピート17,18と同様に、二成分調節系15,19と膣上皮のグローバルな転写応答GBS 19。しかし、完全に膣路内にホスト微生物の相互作用を解明するために、堅牢な動物モデルが必要です。初期の研究は、GBSは、接種したマウス20,21およびラットの膣管から回収することができることを実証しました&lt;SUP&gt;両方の妊娠と非妊娠状態の22。 2005年には、短期的なGBS膣植民地化は、24時間の期間23にわたって膣GBSを治療するためのファージ溶菌酵素の有効性を調べるために、マウスでモデル化しました。数年後、長期GBS膣植民地化のマウスモデルは、GBSの永続性を管理するホストおよび細菌の要因を研究するために開発されました。このモデルは、表面付属17,18およびGBS 2成分系19,24を含むコロニー形成に貢献する数々のGBS因子を同定しました。このモデルは、ホスト応答機構19,25の同定に貢献してきたし、免疫調節ペプチド26とプロバイオティクス27を含むいくつかの治療戦略をテストするために使用されました。このプロトコルは、マウスの膣管内にGBSを接種し、その後コロニー形成を追跡し、さらなる分析のためのサンプルを収集するために必要な指導を提供します。

<table><tbody><tr><td>Sesame oil&amp;nbsp;</td><td>Sigma Aldrich</td><td>S3547-250ML</td><td></td></tr><tr><td>&amp;beta;-Estradiol&amp;nbsp;</td><td>Sigma Aldrich</td><td>E8875-1G</td><td>CAUTION: Wear appropriate PPE. &amp;beta;-estradiol can be absorbed through the skin and mucosal surfaces.&amp;nbsp;</td></tr><tr><td>200 &amp;mu;l gel loading pipette tips&amp;nbsp;</td><td>USA Scientific</td><td>1252-0610</td><td></td></tr><tr><td>Urethro-genital, sterile, calcium alginate swabs</td><td>Puritan</td><td>25-801 A 50</td><td></td></tr><tr><td>CHROMagar StrepB</td><td>DRG International</td><td>SB282</td><td></td></tr><tr><td>Todd Hewitt Broth</td><td>Hardy Diagnostics</td><td>7161C</td><td></td></tr><tr><td>18 G, 1.5 inch needles</td><td>BD</td><td>305199</td><td></td></tr><tr><td>26 G, 0.5 inch needles</td><td>BD</td><td>305111</td><td></td></tr><tr><td>10 ml syringes</td><td>BD</td><td>309604</td><td></td></tr><tr><td>1 ml syringes</td><td>BD</td><td>309659</td><td></td></tr><tr><td>0.45 &amp;mu;m PVDF syringe filters</td><td>Whatman</td><td>6900-2504</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Salt Solution 1x</td><td>Corning</td><td>21-031-CV</td><td></td></tr></tbody></table>

a murine model of group b streptococcus vaginal colonization

成人の30％ - ストレプトコッカス・アガラクティエ （B群連鎖球菌 、GBS）は、消化器および10の膣管のグラム陽性、無症候性植民です。新生児、妊婦、高齢者を含む免疫無防備状態の個体では、GBSは、敗血症、関節炎、肺炎、および髄膜炎を引き起こす侵襲性の病原体に切り替えることができます。 GBSは、新生児の主要な細菌性病原体であるため、現在の予防は、GBS膣コロニー形成およびGBS陽性の母親のその後の周産期抗生物質による治療のための妊娠後期のスクリーニングから構成されています。ヘビーGBS膣の負担が新生児疾患と植民地化の両方の危険因子です。残念ながら、少しはホストと促進またはGBS膣コロニー形成を可能にする細菌の要因について知られています。このプロトコルは、細菌ロアを決定するために、単一のβエストラジオール前処置と毎日のサンプリングを使用して永続的なGBS膣コロニー形成を確立するための技術を説明しますD。また、さらに詳細な方法関心の追加の治療薬または試薬を投与すると、膣洗浄液および生殖管の組織を収集します。このマウスモデルは、妊娠中の母体の膣コロニー形成を制御し、脆弱な新生児への送信を防止するために、潜在的な治療標的につながる膣環境内GBS-ホスト相互作用の理解を促進します。また、女性の膣管内の一般的な細菌宿主相互作用の我々の理解を高めるために対象としています。

During the development of this model, multiple observations were made regarding factors that affect the duration of GBS vaginal colonization. To determine how estrous stage at inoculation impacts GBS bacterial persistence, mice were staged on the day of inoculation via vaginal lavage fluid. Figure 1 illustrates the four stages of the mouse estrous cycle, as determined by wet-mount vaginal lavage fluid, a well-established method29. Mice were divided into groups ...

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

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

グループBのマウスモデル<em&gt;連鎖球菌</em&gt;膣植民地化

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

A Murine Model of Group B Streptococcus Vaginal Colonization

14.2K ビュー.  University of California San Diego School of Medicine. The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

ビデオ: A Murine Model of Group B Streptococcus Vaginal Colonization

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.

の実験的マウスモデルにおけるプッシー接種とサンプル採取のためのプロトコルカンジダ膣炎

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

免疫・感染学

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.

肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

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

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

微小血管系を対象に細菌感染を研究するためのヒト化マウスモデル

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.

Animal models have proven to be invaluable tools in defining host and pathogen specific mechanisms that contribute to the development of chronic inflammation. Here we describe a mouse model of oral infection with the human pathogen Porphyromonas gingivalis and detail methodologies to assess the progression of inflammation at local and systemic sites.

局所および全身所における病原体により誘導される慢性炎症のためのマウスモデル

Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

神経病原の非侵襲的モデル<em&gt;エシェリヒア·コリ</em&gt;新生ラットにおける感染

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, termed chorioamnionitis. Chorioamnionitis exposes the developing fetus to a high risk of organ injuries, perinatal morbidity, and mortality, as well as life-long neurobehavioral impairments and other non-neurological developmental issues. The two most frequent subtypes of GBS isolates from maternal and fetal tissues are serotypes Ia (13%-23%) and III (25%-53%). Our lab has developed and characterized a rat model of GBS-induced chorioamnionitis to study subsequent impacts on the central nervous system of the developing fetus and to understand underlying mechanistic aspects. This article presents the design as well as uses of the preclinical rat model, which closely reproduces the hallmark of GBS-induced chorioamnionitis in humans. This article aims to help scientists reproduce the experimental design as well as to provide support through examples of troubleshooting. The present model may also contribute to potential discoveries through uncovering causes, mechanisms, and novel therapeutic avenues, which remain unsettled in many developmental impairments arising from chorioamnionitis. Furthermore, the use of this model may be extended to the studies of perinatal non-neurological common and severe morbidities affecting, for instance, the retina, bowel, lung, and kidney. The main interest of this research is in the field of GBS-induced fetal neurodevelopmental impairments such as cerebral palsy (CP), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). The rationale supporting this model is presented in this article, followed by procedures and results.

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus GBS

The goal of this protocol is to describe a preclinical animal model of Group B Streptococcus (GBS)-induced chorioamnionitis. The study is designed to investigate mechanistic processes, potential causal links with developmental impairments, and finally to develop translational anti-inflammatory placento- and neuro-protective treatments.

Group B Streptococcus (GBS) is one of the most common bacteria isolated during human pregnancy. It is a leading cause of placental infection/inflammation, ...

膣マイクロバイオームを研究するためのマイクロ流体膣チップ

Modeling Healthy and Dysbiotic Vaginal Microenvironments in a Human Vagina-on-a-Chip

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy reproductive system may lead to life-threatening diseases, infertility, or adverse outcomes during pregnancy. One barrier in the field is that there has been a dearth of preclinical, experimental models that faithfully mimic the physiology and pathophysiology of the FRT. Current in vitro and animal models do not fully recapitulate the hormonal changes, microaerobic conditions, and interactions with the vaginal microbiome. The advent of Organ-on-a-Chip (Organ Chip) microfluidic culture technology that can mimic tissue-tissue interfaces, vascular perfusion, interstitial fluid flows, and the physical microenvironment of a major subunit of human organs can potentially serve as a solution to this problem. Recently, a human Vagina Chip that supports co-culture of human vaginal microbial consortia with primary human vaginal epithelium that is also interfaced with vaginal stroma and experiences dynamic fluid flow has been developed. This chip replicates the physiological responses of the human vagina to healthy and dysbiotic microbiomes. A detailed protocol for creating human Vagina Chips has been described in this article.

著者スポットライト:膣チップを使用した膣マイクロバイオーム相互作用の研究に革命を起こす

This article describes a protocol for creating a microfluidic vagina-on-a-chip (Vagina Chip) culture device that enables the study of human host interactions with a living vaginal microbiome under microaerophilic conditions. This chip can be used as a tool to investigate vaginal diseases as well as to develop and test potential therapeutic countermeasures.

ヒト膣チップにおける健康および嚥下障害の膣微小環境のモデリング

Women's health, and particularly diseases of the female reproductive tract (FRT), have not received the attention they deserve, even though an unhealthy ...

グループBのマウスモデル

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肺炎球菌による鼻腔内コロニー形成時の炎症反応の特徴

微小血管系を対象に細菌感染を研究するためのヒト化マウスモデル

局所および全身所における病原体により誘導される慢性炎症のためのマウスモデル

神経病原の非侵襲的モデル

マウスにおけるガードネレラ膣膀胱曝露によって引き起こされる再発性大腸菌尿路感染症

C-section of Preclinical Animal Model of Chorioamnionitis Triggered by Group B Streptococcus (GBS)

ヒト膣チップにおける健康および嚥下障害の膣微小環境のモデリング

ヒト膣チップにおける健康および嚥下障害の膣微小環境のモデリング

ヒト膣チップにおける健康および嚥下障害の膣微小環境のモデリング