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...

Streptococcus agalactiae, group B Streptococcus (GBS), is an encapsulated, Gram-positive bacterium which is frequently isolated from the gut and genitourinary tract of healthy adults. In the 1970s, GBS emerged as the leading agent of infectious neonatal mortality, with over 7,000 cases of neonatal disease annually1. Early-onset GBS disease (EOD) occurs in the first hours or days of life, arises as pneumonia or respiratory distress, and often develops into sepsis, whereas late-onset disease (LOD) ensues after several months and presents with bacteremia, which frequently advances to meningitis2. As of 2002, the Centers for Disease Control and Prevention recommends universal screening for GBS vaginal colonization in late gestation and intrapartum antibiotic prophylaxis (IAP) to GBS-positive mothers1. Despite the reduction of early-onset disease to approximately 1,000 cases in the United States annually due to IAP, GBS remains the leading cause of early-onset neonatal sepsis, and late-onset occurrence remains unaffected1. Whether in utero, during labor, or even in late-onset cases, neonatal exposure to GBS requires survival, transversal through a number of host environments and barriers, immune evasion, and, in the case of meningitis, crossing of the highly regulated blood-brain barrier2. Upstream of these virulent interactions within the neonate is the initial colonization of the maternal vaginal tract. Maternal GBS vaginal colonization rates range from 8-18% in developed and developing countries, with an estimated average rate of 12.7%3,4. GBS colonization of the vaginal tract during pregnancy may be constant, intermittent, or transient in nature among individual women5. Interestingly, a maternal age &#62; 36 years is associated with persistent colonization6. Numerous biological and socio-economical risk factors for GBS vaginal colonization have been identified. Biological factors include gastrointestinal GBS colonization and absence of Lactobacillus within the gut. However, ethnicity, obesity, hygiene, and sexual activity have also been associated with GBS vaginal carriage7.
Although notorious for causing neonatal infections, GBS also causes a variety of maternal infections both peripartum and postpartum. GBS carriage is increased in women presenting with vaginitis8 and, in some cases, may even be the disease entity9. Additionally, GBS ascension of the reproductive tract during pregnancy may result in intra-amniotic infection or chorioamnionitis10. Moreover, in up to 3.5% of pregnancies, GBS disseminates to the urinary bladder to cause a urinary tract infection or asymptomatic bacteriuria11. GBS bacteriuria during pregnancy is associated with an increased risk of intrapartum fever, chorioamnionitis, preterm delivery, and premature rupture of membranes12. Taken together, the presence of GBS within the vaginal tract is linked to infections of multiple host tissues, and the ability to eliminate GBS from this niche is imperative for both maternal and neonatal health.
Until recently, the majority of work examining GBS interactions with the cervicovaginal tract was limited to in vitro cell models13-15. These in vitro experiments have revealed bacterial factors that are important for adherence, including surface proteins such a pili and serine-rich repeats17,18, as well as two-component regulatory systems15,19 and the global transcriptional response of the vaginal epithelium to GBS19. However, to fully elucidate the host-microbe interactions within the vaginal tract, a robust animal model is necessary. Early work demonstrated that GBS can be recovered from the vaginal tract of inoculated mice20,21 and rats22 in both pregnant and non-pregnant conditions. In 2005, short-term GBS vaginal colonization was modelled in mice to examine the efficacy of a phage lytic enzyme to treat vaginal GBS over a 24 hr period23. Several years later, a long-term GBS vaginal colonization mouse model was developed to study host and bacterial factors governing GBS persistence. This model has identified numerous GBS factors contributing to colonization, including surface appendages17,18 and GBS two-component systems19,24. This model has contributed to the identification of host response mechanisms19,25 and was used to test several therapeutic strategies, including immunomodulatory peptides26 and probiotics27. This protocol gives the necessary guidance to inoculate GBS into the mouse vaginal tract and to subsequently track colonization and collect samples for further analyses.

<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

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

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.

A Murine Model of Group B Streptococcus Vaginal Colonization

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

Research

JoVE Journal

Immunology and Infection

14.3K Views.  University of California San Diego School of Medicine.  Het doel van dit protocol is voor de menselijke groep B Streptococcus (GBS) vaginale kolonisatie na te bootsen in een muismodel. Deze werkwijze kan worden gebruikt om immuunreacties van de gastheer en bacteriële factoren die GBS vaginale persistentie te onderzoeken, alsmede therapeutische strategieën testen. 

Video: 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.

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

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.

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.

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.

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.

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.

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, ...

Microfluidic Vagina Chip for Studying the Vaginal Microbiome

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.

Author Spotlight: Revolutionizing Research on Vaginal Microbiome Interactions Using a Vaginal Chip

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.

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

Bioengineering

Models of Murine Vaginal Colonization by Anaerobically Grown Bacteria

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four women experience bacterial vaginosis, in which a low level of lactobacilli is accompanied by an overgrowth of diverse anaerobic bacteria. This condition has been associated with many health complications, including risks to reproductive and sexual health. While there is growing evidence showing the complex nature of microbial interactions in human vaginal health, the individual roles of these different anaerobic bacteria are not fully understood. This is complicated by the lack of adequate models to study anaerobically grown vaginal bacteria. Mouse models allow us to investigate the biology and virulence of these organisms in vivo. Other mouse models of vaginal bacterial inoculation have previously been described. Here, we describe methods for the inoculation of anaerobically grown bacteria and their viable recovery in conventionally raised C57Bl/6 mice. A new, less stressful procedural method for vaginal inoculation and washing is also described. Inoculation and viable recovery of Gardnerella are outlined in detail, and strategies for additional anaerobes such as Prevotella bivia and Fusobacterium nucleatum are discussed.

The protocol presents a mouse model of vaginal colonization with anaerobically cultured human vaginal bacteria. We focus on Gardnerella vaginalis,&#160;while including suggestions for Prevotella bivia and Fusobacterium nucleatum. This protocol can also be used as a guide for vaginal inoculations and viable recovery of other anaerobically grown bacteria.

The mammalian vagina can be colonized by many bacterial taxa. The human vaginal microbiome is often dominated by Lactobacillus species, but one-in-four ...

Isolation of Splenic Microvesicles in a Murine Model of Intraperitoneal Bacterial Infection

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular effectors. In rats, spleen MVs (SMVs) are surrogate markers of pathophysiological conditions. Previous in vitro studies demonstrated that Porphyromonas gingivalis (P. gingivalis), a major periodontal pathogen, enables&#160;the endothelial shedding and apoptosis while lipopolysaccharide (LPS) favors the shedding of splenocyte-derived microvesicles (SMVs). In vivo studies showed the feasibility of pharmacological control of SMV shedding. The present protocol establishes a standardized procedure for isolating splenic SMVs from the P. gingivalis acute murine infection model.&#160;P. gingivalis infection was induced in young C57BL/6 mice by intraperitoneal injection (three injections of 5 x 107 bacteria/week). After two weeks, the spleens were collected, weighed, and the splenocytes were counted. SMVs were isolated and quantified by protein, RNA, and prothrombinase assays. Cell viability was assessed by either propidium iodide or trypan blue exclusion dyes. Following splenocyte extraction, neutrophil counts were obtained by flow cytometry after 24 h of splenocyte culture. In P. gingivalis-injected mice, a 2.5-fold increase in spleen weight and a 2.3-fold rise in the splenocyte count were observed, while the neutrophils count was enhanced by 40-folds. The cell viability of splenocytes from P. gingivalis-injected mice ranged from 75%-96% and was decreased by 50% after 24 h of culture without any significant difference compared to unexposed controls. However, splenocytes from injected mice shed higher amounts of MVs by prothrombinase assay or protein measurements. The data demonstrate that the procoagulant SMVs are reliable tools to assess an early spleen response to intraperitoneal&#160;P. gingivalis infection.

Microvesicles shed from the plasma membrane act as cellular effectors. Spleen microvesicles (SMVs) are surrogate markers of pathophysiological conditions. In rats and mice, SMV content and properties characterize aging or inflammation and are altered by cytoprotective treatments, making them a valuable yet abundant monitoring tool for preclinical research.

Microvesicles (MVs) are submicron fragments released from the plasma membrane of activated cells that act as proinflammatory and procoagulant cellular ...

A Murine Model of Group B Streptococcus Vaginal Colonization

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