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

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

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.</l...

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.

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			<td style="width:119px;">E8875-1G</td>
			<td>CAUTION: Wear appropriate PPE. &#946;-estradiol can be absorbed through the skin and mucosal surfaces.&#160;</td>
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			<td height="21" style="height:21px;width:216px;">18 G, 1.5 inch needles</td>
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			<td height="21" style="height:21px;width:216px;">26 G, 0.5 inch needles</td>
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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 ...

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