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 demonstrates robust reproducibility, with modest inter-experimental variability in both the length of GBS persistence and the bacterial burden. The representative results demonstrated in this study are the compilation of independent experiments and should be a reference for future experimental design; however, direct comparisons across GBS strains and mouse lines should be made with care.
This model mimics human colonization in that mucosal vaginal GBS colonization of mice appears to be restricted to the reproductive tract. Although ascension into the cervix and uterus has been observed with multiple GBS strains25, mice do not display signs of morbidity or mortality, even after months of colonization. Furthermore, depending on the GBS and mouse strains studied, mice display consistent or transient vaginal colonization, which is useful for studying bacterial factors and host immune responses, respectively. In this model, some mice display intermittent colonization; it is currently unknown whether mice become recolonized at later time points or if colonization falls below the limit of detection, typically 50 to 100 CFU, at certain time points. Of note, transmission between mice has not been observed when colonized and non-colonized mice are housed together over several weeks (data not shown).
This method uses a commercially-available selective and differential medium for GBS to quantify mouse bacterial burdens. GBS grows as bright pink or mauve colonies that are readily visible after 24 hr of incubation. This media has been shown to have higher sensitivity for detecting GBS compared to other media, including blood agar and Granada medium32, and has been used in combination with latex bead agglutination tests to confirm GBS clinical isolates18. In this study, bright pink or mauve colonies from non-GBS colonized mice have never been recovered. Some endogenous flora, typically Enterococcus species, will grow as blue colonies, and some colonies will appear white, grey, or very pale pink. Importantly, plates should be counted after 18 to 24 hr of incubation, as non-GBS colonies may incorporate the pink pigment if left in the incubator or on the benchtop for longer periods. We have observed, as has been reported previously32, that some S. pyogenes isolates will form pink colonies on CHROMagar StrepB, but these colonies are typically smaller than GBS colonies. S. pyogenes has not been isolated from the endogenous vaginal flora of mice in these studies, but this observation should be considered in future work.
The majority of results were obtained from the outbred CD-1 mouse line, which demonstrates robust innate immune responses within the first few days post-inoculation, with subsequent bacterial clearance in the majority of mice19. Others have also tested additional GBS strains and have observed longer persistence times33,34. Since the development of this model, other groups have begun to develop similar mouse models of GBS vaginal colonization to examine the impact of host immune responses33,35, preventative therapies36,37, and transmission to the fetus in utero34,38. These differences may be explained by a variety of factors, including genetic determinants that impact immune responses and the composition of native vaginal flora. We have compiled a list of the GBS strains and respective mouse lines that have been studied to date in GBS vaginal colonization studies (Table 1). The number of recent studies with animal models highlights the interest and necessity of these types of models within the field of GBS pathogenesis research.
Within the vaginal tract, mucosal immunity is tightly regulated by steroid hormones44, and even one dose of &#946;-estradiol, as described in this model, perturbs the host immune response. Even so, there are robust innate immune responses within the first few days of GBS colonization19,25, suggesting that affected immune responses are largely intact. Models that involve repeated &#946;-estradiol injections may prolong GBS vaginal persistence, as demonstrated in this study (Figure 3) and by others33. However, immune responses and reproductive tract physiology may be more confounded, making results difficult to interpret. Importantly, the differences described across different GBS isolates and mouse strains in this study may be altered during models of sustained estrus and should be investigated in future work. Of note, neither estrus stage nor GBS serotype impacted vaginal colonization in a rat model22. Additionally, the human acidic vaginal pH of 3.6 to 4.545 drastically differs from the more neutral murine vaginal pH of 6.546, which may impact GBS gene expression and subsequent factors contributing to colonization. Lastly, native vaginal flora is distinct between humans and murine model counterparts, and future work should examine GBS colonization in gnotobiotic mice carrying human vaginal flora.
In summary, these studies have sought to examine host and bacterial factors that govern GBS vaginal colonization. Primarily, the robust, innovative animal model of GBS vaginal colonization developed in this work can be utilized to describe complex host-microbe interactions in an in vivo vaginal environment. The information obtained from this model has already greatly increased the knowledge of host immune components and specific GBS genes that control GBS vaginal persistence. Moreover, these results have raised additional questions and will be useful for further development of novel therapeutics to limit maternal GBS vaginal colonization and subsequent exposure of the newborn.

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 border="0" cellpadding="0" cellspacing="0" width="592">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="43">
			<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CHROMagar StrepB</td>
			<td style="width:91px;">DRG International</td>
			<td style="width:119px;">SB282</td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</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 based on this initial stage, and GBS persistence was monitored over time via vaginal swabbing. 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 (Figure 2). Based on these results, in the current model, mice are treated with &#946;-estradiol one day prior to GBS inoculation to synchronize them into the proestrus stage.
Other murine models of reproductive tract infections have demonstrated an increased ability of the pathogenic organism to persist when mice are sustained in the estrus stage through exogenous estradiol treatment30,31. To determine if this phenomenon also occurred during vaginal colonization with GBS, GBS persistence was monitored during repeated &#946;-estradiol treatment. Sustained estrus promoted GBS A909 (American Type Culture Collection, ATCC #BAA-1138) persistence in CD-1 mice, with 90% colonization 2 weeks post-inoculation (Figure 3A). In a typical experiment with one dose of &#946;-estradiol prior to inoculation, only 40-50% of mice were colonized one week post-inoculation (Figure 4). The mean GBS CFU recovered from these mice mimics the percentage of colonization (Figure 3B). Although these results were obtained from independent experiments, these data demonstrate that maintaining continuous estrus promotes GBS vaginal persistence in the majority of CD-1 mice.
While conducting colonization experiments using different human GBS isolates, it was observed that strains varied in their ability to persist in CD-1 mice, ranging from several days to beyond a month. Of the strains tested, NCTC 10/84 had the shortest duration, A909 and COH1 persisted for one to two weeks, whereas strain CJB111 persisted in the majority of mice for two weeks (Figure 4) and even beyond a month (data not shown). To date, there has been no observed correlation of serotype and ability to persist in the mouse vaginal tract; however, significant differences between individual GBS strains have been reported25. The ability of GBS to colonize multiple inbred and outbred mouse lines was also examined. GBS strain A909 persisted in the vaginal tract for approximately one week in outbred CD-1 mice and inbred FVB mice (Figure 5). Alternatively, the majority of inbred BALB/c and C57BL/6 were colonized at one week (Figure 5) and remained colonized for a month or beyond (data not shown).
Using this protocol, GBS vaginal colonization in vivo was visualized by inoculating mice with a plasmid GFP-expressing GBS strain and collecting tissue for fluorescent microscopy. GFP-GBS was detected both adhering to murine vaginal epithelium (Figure 6A) and in close proximity to other native vaginal flora (Figure 6B). No GFP signal was detected in mice that had cleared the GFP-GBS at the time of tissue collection (data not shown).
<img alt="Figure 1" src="/files/ftp_upload/54708/54708fig1.jpg" /> 
Figure 1: Identifying the Stage of Estrus from Unstained Murine Vaginal Lavage Fluid. (A) Proestrus: abundant nucleated squamous epithelial cells (black arrows). (B) Estrus: abundant cornified squamous epithelial cells (blue arrows). (C) Metestrus: mixture of nucleated and cornified squamous epithelial cells and predominantly leukocytes (grey arrows). (D) Diestrus: abundant leukocytes. Magnification = 100X, scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54708/54708fig2.jpg" /> 
Figure 2: Estrous Stage Impacts Vaginal Persistence of GBS. Percent of CD-1 mice colonized with 1 &#215; 107 CFU GBS A909 over time. Mice were grouped (n = 7 - 11/group) based on estrous stage at the time of GBS inoculation, as determined by vaginal lavage fluid. One independent experiment is shown. This figure has been modified from previously-published work and reprinted with permission19. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54708/54708fig3.jpg" /> 
Figure 3: Continued Treatment with &#946;-estradiol Promotes GBS Vaginal Persistence. Percent colonized (A) or recovered CFU (B) of CD-1 mice (n = 10) inoculated with 1 &#215; 107 CFU GBS A909 and maintained on &#946;-estradiol treatment. Mice were injected with &#946;-estradiol one day prior to GBS inoculation and on days 1, 3, and 5 post-inoculation. One independent experiment is shown. The line in (B) represents mean recovered CFU. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54708/54708fig4.jpg" /> 
Figure 4: GBS Strains Differ in Their Ability to Persist in the Vaginal Tract. Outbred CD-1 mice (n = 10/group) were injected with a single dose of &#946;-estradiol one day prior to GBS inoculation with 1 &#215; 107 CFU of the given GBS strains. Experiments with each strain were carried out independently and were repeated at least three times; one representative result is shown. This figure has been modified from previously-published work and reprinted with permission25. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54708/54708fig5.jpg" /> 
Figure 5: Mouse Strains Differ in Their Ability to Be Colonized with GBS in the Vaginal Tract. Mice from indicated background strains were injected with a single dose of &#946;-estradiol one day prior to inoculation with 1 &#215; 107 CFU of GBS A909. Experiments with each strain were carried out independently and were repeated at least twice; one representative result is shown. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54708/54708fig6.jpg" /> 
Figure 6: Fluorescent Imaging of GBS within the Mouse Vaginal Tract. Visualization of GFP- expressing GBS (green) along the vaginal epithelium at magnification = 630X, scale bar = 50 &#181;m (A), and in close proximity to endogenous vaginal microbes at magnification = 1,000X, scale bar = 20 &#181;m (B). Blue stain = DAPI. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54708/54708fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/54708/54708table1.jpg" /> 
Table 1: Compilation of GBS Strains and Mouse Lines Utilized for Vaginal Colonization Studies. GBS background strains, mouse lines, and dosage of &#946;-estradiol are indicated. Studies using the model described in this work are highlighted in grey.

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

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

14.1K Views.  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.

Video: 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.

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.

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.

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

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.

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.

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

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 ...

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.

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.

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.

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.

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