The studies were supported in part by PRIN 2012 [grant number 2012WJSX8K]: &ldquo;Host-microbe interaction models in mucosal infections: development of novel therapeutic strategies&rdquo; and by PRIN 2017 [2017SFBFER]: &ldquo;An integrated approach to tackle the interplay among adaptation, stressful conditions and antimicrobial resistance of challenging pathogens&rdquo;.

This protocol was conducted to minimize animal suffering and reduce the number of mice in accordance with the European Communities Council Directive of November 24, 1986 (86/609/EEC). In vivo experiments reported in this study were approved by the Ethical Animal Care and Use Committee (Prot. number 2, 14 December 2012) and the Italian Ministry of Health (Prot. number 0000094-A-03/01/2013). All the procedures should be performed inside the Biosafety Cabinet 2 (BSC2) in a BSL2 room, and the potential infected waste should be disposed in dedicated containers.
1. Infection of Mice with N. meningitidis Serogroup C Strain
CAUTION: N. meningitidis is potentially a harmful pathogen and all necessary precautions must be taken when handling this microorganism. The entire experimentation requires Biosafety Level 2 (BSL2) containment. The researcher involved in animal studies should wear disposable personal protective equipment (PPE) for the duration of the experiment.
<ol>
	<li>Preparation of bacteria for in vivo infection studies
	<ol>
		<li>Pick a single colony from a fresh Neisseria meningitidis culture on GC (Gonococcal) agar plate supplemented with 1% (vol/vol) Polyvitox supplement and inoculate in 10 mL of GC broth.</li>
		<li>Grow the bacteria at 37 &#176;C in an orbital shaker incubator with the speed of 220 rpm. Keep checking the O.D. of the culture with a spectrophotometer. Grow the culture until the early exponential phase at an optical density OD600nm of 0.7, corresponding to &#8776; 7 x 108 CFU/mL.</li>
		<li>Once the required O.D. is obtained, make frozen stocks by adding 10% glycerol. Dispense 1 mL of the culture in the cryovials. Store the vials at -80 &#176;C until use. 
		NOTE: Although it is better to use fresh bacterial culture, frozen stocks were used to simplify and standardize the in vivo experiment. Usually the frozen stocks have been employed within approximately 6 months from the preparation.</li>
		<li>Before the infection, thaw frozen bacteria at room temperature.</li>
		<li>Harvest the bacterial cells by centrifugation for 15 min at 1,500 x g and resuspend in 1 mL of fresh GC broth containing iron dextran (5 mg/kg). 
		NOTE: The GC broth is prepared with the addition of iron dextran (5 mg/kg) in order to favor the replication of meningococci in the host tissue14,18,27.</li>
		<li>Before using, perform viable counts of bacteria to determine the exact number of CFU for infection. To do so, pick up 10 &#181;L of bacterial suspension and proceed with serial dilutions and spread each dilution on GC agar plates and incubate at 37 &#176;C with 5% CO2, for 18-24 h.</li>
	</ol>
	</li>
	<li>Intra-cisternal injection of mice 
	NOTE: The entire procedure is performed in the laminar flow cabinet to maintain aseptic conditions.
	<ol>
		<li>House laboratory mice (8 week-old, female Balb/c) under specific pathogen free conditions. Provide food pellets and water ad libitum.</li>
		<li>Settle the animals in the new environment for 1 week before starting the experiment.</li>
		<li>Before starting the experiment, weigh and evaluate their body temperature. 
		NOTE: The inbred Balb/c female mice of eight-week-old weigh an average of about 19 g. The average temperature of laboratory mice physiologically ranging from 36-38 &#176;C24.</li>
		<li>Scruff the animal from the neck,&#160;clean the abdomen area with the 70% ethanol&#160;and inject i.p. iron dextran (dissolved in 1 % phosphate saline buffer, 250 mg/kg) in the lower right quadrant of the murine abdomen by using a 25 G needle 0.5 mm x 16 mm, approximately 2-3 h before the infection. 
		NOTE: The intraperitoneal injection was performed in the lower right quadrant of the murine abdomen to avoid damaging abdominal organs such as urinary bladder, cecum, etc. The administration of exogenous iron source, in the form of iron dextran, to animals prior to the infection favors the bacterial multiplication in the host14,18,27.</li>
		<li>After 2-3 h, perform animal anesthesia with ketamine (50 mg/kg) and xylazine (3 mg/kg) and ophthalmic lubricant.</li>
		<li>Check for the depth of anesthesia by ensuring the absence of pain response upon pinching the toe.</li>
		<li>Position the mouse in sternal decubitus and carefully stretch the limbs and the cervical spine to keep the vertebral column in a straight position.</li>
		<li>Gently mix the bacterial suspension to maintain a consistent suspension before loading a syringe of 30 G needle x 8 mm. 
		NOTE: Prepare the bacterial suspension as close as possible to the injection time; meanwhile, store it at room temperature.</li>
		<li>Based on the post thawing bacterial titer (CFU/mL), proceed with the calculation of the total CFU to be used, with respect to the total number of animals to be infected (CFU bacterial dose per number of animals). Proceed with the calculation of the exact volume to be taken from the vial to obtain the total CFU applying a proportion between the post thawing bacterial titer (CFU/mL) and the total CFU useful for the infection of n mice (post thawing bacterial titer CFU: ml = total CFU : x). Establish the final volume with respect to the total number of animals to be infected. 
		NOTE: In these experiments, a wide range of titer from 104 to 109 per animal was used.</li>
		<li>Clean the surgical area with 70% Ethanol.</li>
		<li>Identify the injection point with the help of a needle and inject the established CFU of meningococci (wild type strain and isogenic mutant strain), or GC broth supplemented with iron dextran (5 mg/kg) as control, in a total volume of 10 &#181;L into the cisterna magna of mice through an occipital burr hole by using a 30 G needle x 8 mm.
		<ol>
			<li>Perform the cisternal inoculum by placing the needle at the craniocervical junction, specifically in the dorsal subarachnoid space. Ventroflex the head to make this space accessible28.</li>
			<li>Briefly, place the animal in lateral recumbency, hold the ears out of the way and flex the neck moderately (90 to 100&#176;). Ensure that the midline of the neck and the head (from the nose to the occiput) are in perfectly parallel position to the tabletop.</li>
			<li>Touch the atlas wings and make sure that they overlap, eliminating axial rotation. A natural indentation can usually be touched on midline where the needle is most likely to enter the occipital hole.</li>
			<li>Discard the syringe and needle safely after the injection of mice with Neisseria.</li>
		</ol>
		</li>
		<li>Place the animal in the cage and wait for the awakening and full recovery of movement.</li>
		<li>Keep the cages with infected mice under a laminar flow cabinet.</li>
		<li>Monitor mice, 24 h post infection, for clinical signs of coma according to the coma scale29. Coma scale: 1 = coma, 2 = does not stand upright after being turned on the back, 3 = stands upright within 30 s, 4 = stands upright within 5 s, minimal ambulatory activities, 5 = normal. 
		&#160;NOTE:&#160;When in animals was recorded pain, analgesia with meloxicam (5 mg/kg i.p. for duration of study) was administered.</li>
		<li>Proceed for the animal survival or CFU counts assay on the infected animals as detailed in step 2.</li>
		<li>Perform euthanasia of mice with a score of 2 by cervical dislocation and record as dead for statistical analysis.</li>
	</ol>
	</li>
</ol>
2. Animal Survival and CFU Counts
<ol>
	<li>Animal survival
	<ol>
		<li>Prepare bacterial inocula at different doses (ranging from 104 to 109 CFU per mouse) to infect animals by the i.cist. route (see step 1.2).</li>
		<li>Inoculate control mice with GC broth, supplemented with iron dextran (5 mg/kg), in the same manner.</li>
		<li>Monitor animals for clinical symptoms: ruffled fur, hunched appearance, hypothermia, weight loss, lethargy, or moribund24,25,26,30, every day throughout the whole experiment for 168 h (7 days)&#160;at least twice a day for the duration of the experiment.</li>
		<li>Measure the body weight and temperature by using a digital balance and a rectal thermometer, respectively everyday.</li>
		<li>Record the survival of mice for a week. 
		NOTE: Record the natural death of animal post infection while the animals that reach a coma value of 2 or that survive over 168 h of observation will be euthanized.</li>
		<li>Anesthetize mice with a coma value of 2 or that survive over the observation time with ketamine (50 mg/kg) and xylazine (3 mg/kg) and apply ophthalmic ointment.</li>
		<li>Check that the pain response is absent by toe pinch.</li>
		<li>Perform euthanasia of mice by cervical dislocation and record as dead for statistical analysis.</li>
	</ol>
	</li>
	<li>Evaluation of colony forming units (CFU) counts in peripheral organs
	<ol>
		<li>Use a sub-lethal bacterial dose (5 x 105CFU/mice) on the basis of animal survival results to inoculate animals by the i.cist. route (see subsection 1.2).</li>
		<li>Monitor the rectal temperature every day during the infection and perform anesthesia of animals at 48 h post infection as mentioned in step 2.</li>
		<li>Proceed with the 70% ethanol disinfection of the chest and withdraw 600-700 &#181;L of blood by cardiac puncture of the chest cavity using a 25 G needle 0.5 mm x 16 mm.</li>
		<li>Collect the blood in a tube containing 3.8% sodium citrate and store at -80 &#176;C for the later viable bacterial cell counts.</li>
		<li>Perform cervical dislocation to sacrifice the animals. Confirm the death recording the absence of heartbeat, after sacrificing the mouse according to all relevant institutional and ethical guidelines.</li>
		<li>Lay the mouse in the supine position and use scissors and forceps to proceed with the cut of the fur along the sagittal plane of body. Fix the skin with the fur on the sides of the body with pins.</li>
		<li>Cut off the peritoneal membrane using sharp scissors. Use scissors and disposable forceps to excise the organs (e.g., spleen and liver) and put each one in sterile Petri dish with 1 mL of GC broth supplemented with 10% (vol/vol) glycerol.</li>
		<li>Safely dispose the mouse body as per IACUC guidelines.</li>
		<li>Homogenize the organs mechanically at room temperature with the plunger of a 5 mL syringe for about 2-3 min until a single-cell suspension is formed and transfer it in a tube.</li>
		<li>Put the tube with the homogenized tissue sample immediately on the dry ice. 
		NOTE: Samples can be stored at -80 &#176;C in a 2 mL sterile tube for performing the viable bacterial cell counts evaluation at later points.</li>
		<li>Make GC agar plates with antibiotics when required. Dry the plates prior to the use pre-incubating at 37 &#176;C for 2-3 h.</li>
		<li>Prepare 10-fold serial dilutions of the samples in GC broth from each homogenized tissue and plate onto GC agar plates. Incubate overnight at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
</ol>
3. Preparation of Brain Tissues for CFU Count
<ol>
	<li>Use a sub-lethal bacterial dose (5 x 105&#160;CFU/mice) on the basis of animal survival results to inoculate animals by the i.cist. route (see subsection 1.2).</li>
	<li>Perform anesthesia of animals at established infection time as mentioned in step 2.</li>
	<li>Perform euthanasia of animals by cervical dislocation.</li>
	<li>Clean the surgical area with 70% Ethanol.</li>
	<li>Cut off the head of mouse using a large scissors.</li>
	<li>Resect the fur and the skin with the help of small surgical scissors and a fine tipped steel forceps, proceeding towards the top of the skull to be able to clearly see the sutures and to guide the opening of the cranium.</li>
	<li>Insert the tip of a tiny scissors through the foramen magnum to open the skull.</li>
	<li>Cut towards the center of the cranium and across the midline of parietal bone to the opposite side of the skull. Gently cut along the lateral rim of the lambdoid suture.</li>
	<li>Lift the cranium starting from the posterior parietal corner and pull it diagonally upward to discover the brain, by using fine tipped forceps.</li>
	<li>Be sure that the brain tissue is not attached to any bone of the head, when the skull is lifted.</li>
	<li>Use disposable forceps to remove any connective tissue between the skull and the brain, to ensure that brain tissue being removed along with the skull.</li>
	<li>Do not allow the brain tissue dry out too much. Place the brain, using disposable forceps, in a Petri dish with 1 mL of GC broth supplemented with 10% (vol/vol) glycerol.</li>
	<li>Homogenize the brain mechanically with the plunger of a 5 mL syringe (see step 2.2.9). Transfer the samples into a 2 mL sterile tube and store -80 &#176;C for the later viable bacterial cell counts evaluation as discussed in step 2.2.</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+diagnosis+of+experimental+meningitis+by+bacterial+heat+production+in+cerebrospi-+nal+fluid.">Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospi- nal fluid.</a> BMC Infectious Diseases. 7, 116 (2007).</li><li>Tuomanen, E. I., Saukkonen, K., Sande, S., Cioffe, C., Wright, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+inflammation,+tissue+damage,+and+mortality+in+bacterial+meningitis+in+rabbits+treated+with+monoclonal+antibodies+against+adhesion-promoting+receptors+of+leukocytes.">Reduction of inflammation, tissue damage, and mortality in bacterial meningitis in rabbits treated with monoclonal antibodies against adhesion-promoting receptors of leukocytes.</a> Journal of Experimental Medicine. 170, 959-969 (1989).</li><li>Goldschneider, I., Gotschlich, E. C., Artenstein, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunity+to+the+meningococcus.+I.+The+role+of+humoral+antibodies.">Human immunity to the meningococcus. I. The role of humoral antibodies.</a> Journal of Experimental Medicine. 129, 1307-1326 (1969).</li><li>Goldschneider, I., Gotschlich, E. C., Artenstein, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunity+to+the+meningococcus.+II.+Development+of+natural+immunity.">Human immunity to the meningococcus. II. Development of natural immunity.</a> Journal of Experimental Medicine. 129, 1327-1348 (1969).</li><li>World Health Organization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+methods+for+the+diagnosis+of+meningitis+caused+by+Neisseria+meningitidis,+Streptococcus+pneumoniae,+and+Haemophilus+influenzae:+WHO+manual,+2nd+Edition.">Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae: WHO manual, 2nd Edition.</a> World Health Organization. , (2011).</li><li>Chiavolini, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+inducing+experimental+pneumococcal+meningitis+in+outbred+mice.">Method for inducing experimental pneumococcal meningitis in outbred mice.</a> BMC Microbiolology. 4, 36 (2004).</li><li>Zhang, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracranial+Subarachnoidal+Route+of+Infection+for+Investigating+Roles+of+Streptococcus+suis+Biofilms+in+Meningitis+in+a+Mouse+Infection+Model.">Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model.</a> Journal of Visualized Experiments. (1), e137 (2018).</li><li>Pagliuca, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+of+Bacteroides+fragilis+protection+from+Bartonella+henselae-induced+damage.">Evidence of Bacteroides fragilis protection from Bartonella henselae-induced damage.</a> PLoS One. 7, 49653 (2012).</li><li>Larson, J. A., Howie, H. L., So, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neisseria+meningitidis+accelerates+ferritin+degradation+in+host+epithelial+cells+to+yield+an+essential+iron+source.">Neisseria meningitidis accelerates ferritin degradation in host epithelial cells to yield an essential iron source.</a> Molecular Microbiology. 53, 807-820 (2004).</li><li>Festing, M. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+variability+of+inbred+and+outbred+mice.">Phenotypic variability of inbred and outbred mice.</a> Nature. 263, 230-232 (1976).</li><li>Festing, M. F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Warning:+the+use+of+heterogeneous+mice+may+seriously+damage+your+research.">Warning: the use of heterogeneous mice may seriously damage your research.</a> Neurobiology of Aging. 20, 237-244 (1999).</li></ol>

The authors have nothing to disclose.

In this study, we describe an experimental protocol to induce meningococcal meningitis in adult mice by i.cist. inoculation of meningococcal bacteria. To our knowledge, no other model of meningococcal meningitis has been developed in laboratory mice infected by i.cist. route; in the past, this way has been explored to provide models of meningococcal meningitis in both rat31 and rabbit32. It is well-known that the highest rate of meningococcal disease is found between young ...

Neisseria meningitidis is a Gram negative &#946;-proteobacterium restricted to the human host, well known for being one of the most common causes of meningitis and sepsis in the human population across the world. It colonizes the upper respiratory tract (nose and throat) of healthy and asymptomatic carriers (2-30% of the population), but the bacterium sometimes evades various host immune defenses and spreads from the bloodstream to the brain causing an uncontrolled local inflammation, known as meningococcal meningitis. A combination of host and bacterial factors appears to contribute to the transition from the commensal to the invasive behavior1.
N. meningitidis is specialized exclusively in human colonization and infection. It has a narrow host range and, therefore, has limited in vivo pathogenesis studies due to the lack of suitable animal models that reproduce the human meningococcal disease. As a result, it had led to fundamental gaps in the comprehension concerning the pathogenesis of septicemia and meningitis caused by meningococcus. In the last decades, the development of many in vitro systems allowed the identification of several meningococcal virulence factors2,3,4. Although these valuable studies provided important insights to understand the role of these factors for a successful meningococcal infection, these models did not allow assessment of the consequences of bacterial interactions with the humoral and cellular immune system and even less with the whole tissue. In vivo animal models of infection are of great relevance as well for the evaluation of protection degree conferred by vaccine formulations. As a human-tropic pathogen, meningococcus possess appropriate determinants necessary for successful infection such as surface structures (i.e., type IV pili and opacity proteins) and iron uptake systems for human receptors and transport proteins (i.e., transferrin and lactoferrin)5,6,7 to properly adhere, survive and invade the human host. Finally, the genetic variation abilities of the pathogen to evade and/or block the human immune response further contribute to the high species tropism8,9. Therefore, the absence of specific host factors, involved in the interaction, can block steps of the pathogen&#8217;s life cycle, establishing significant difficulties in the development of small animal models summarizing the meningococcal life cycle.
Over the past decades, several approaches have been developed to improve our understanding of the meningococcal infectious cycle. Infections of two animal model, mouse and rat, either intraperitoneally (i.p.) or intranasally (i.n.), were developed to reproduce meningococcal disease10,11,12,13,14,15,16,17. The laboratory mouse is probably one of the more versatile animals for inducing experimental meningococcal infection.
However, the i.p. way of infection leads to the development of severe sepsis although it does not mimic the natural route of infection, whereas the i.n. route of infection was useful to evaluate meningococcal pathogenesis, even though it may induce lung infection prior to sepsis10,11,12,13,14,15,16,17.
The i.p. mouse model was instrumental to assess the protection from the meningococcal challenge10,11,12. The mouse model of meningococcal colonization based on the i.n. route of infection has been developed with infant mice, as they are more susceptible to meningococci, to reproduce an invasive infection mimicking the course of the meningococcal disease in humans13,14,15,16,17. Moreover, to promote meningococcal replication in the murine host, a growing number of technical strategies were also applied including the administration of the iron to the animals to improve the infection, the use of high bacterial inoculum, mouse-passaged bacterial strain as well as the employment of infant or immunocompromised animal hosts10,13,15,18,19. Expression of specific human factors like CD4620 or transferrin21 has increased the susceptibility of mice to this human-tropic bacterium; the employment of the human skin xenograft model of infection has also been useful to evaluate the adhesion ability of meningococci to human endothelium22,23. Collectively, the recent development of humanized transgenic mice has improved the understanding of the meningococcal pathogenesis and its host interactions.
Previously, we developed a murine model of meningococcal meningitis where the inoculation of bacteria was performed into the cisterna magna of adult mice with mouse-passaged bacteria24. Clinical parameters and the survival rate of infected mice demonstrated the establishment of meningitis with characteristics comparable to those seen in the human host, as well as, the microbiological and histological analyses of the brain. From these infected mice, bacteria were, also, recovered from blood, liver, and spleen, and bacterial loads from peripheral organs correlated with the infectious dose. In particular, this model was employed to evaluate the virulence of an isogenic mutant strain defective in the L-glutamate transporter GltT24. Recently, using our mouse model of meningococcal meningitis based on i.cist. route with serogroup C strain 93/42862,24 and an isogenic mutant defective in cssA gene encoding for UDP-N-acetylglucosamine 2-epimerase25, we have analyzed the role of exposed sialic acid in the establishment of disease in mice.
In this protocol, we describe a straightforward method to induce experimental meningococcal meningitis based on the i.cist. route of infection in Balb/c adult mice. This method is particularly useful for the characterization of meningococcal infection in a murine host, as well as for the assessment of the virulence between wild type reference strains and isogenic mutants. The intra-cisternal route of infection ensures complete delivery of the meningococci directly into the cisterna magna, which in turn facilitates bacterial replication in the cerebrospinal fluid (CSF) and induces meningitis with features that mimic those present in humans2,24,25,26.

<table>
	<tbody>
		<tr>
			<td>1,8 Skirted Cryovial With external thread</td>
			<td>Starlab</td>
			<td>E3090-6222</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml Polypropylene Conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td>30 x 115mm</td>
		</tr>
		<tr>
			<td>Adson Forceps</td>
			<td>F.S.T.</td>
			<td>11006-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Alarm-Thermometer</td>
			<td>TESTO</td>
			<td>9000530</td>
			<td></td>
		</tr>
		<tr>
			<td>BactoTM Proteose Peptone</td>
			<td>BD</td>
			<td>211693</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Micro Fine syringe</td>
			<td>BD</td>
			<td>320837</td>
			<td>U-100 Insulin</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 1ml 25GA 5/8in</td>
			<td>BD</td>
			<td>300014</td>
			<td>05x16mm</td>
		</tr>
		<tr>
			<td>BD Plastipak syringe 5ml</td>
			<td>BD</td>
			<td>308062</td>
			<td>07 x 30mm</td>
		</tr>
		<tr>
			<td>BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET</td>
			<td>Bio Air s.c.r.l.</td>
			<td>Aura B3</td>
			<td></td>
		</tr>
		<tr>
			<td>BioPhotometer</td>
			<td>Eppendorf</td>
			<td>Model #6131</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle D</td>
			<td>Tecniplast</td>
			<td>D</td>
			<td>Graduated up to:400ml, Total Volume 450ml, 72x72x122mm</td>
		</tr>
		<tr>
			<td>C150 CO2 Incubator</td>
			<td>Binder</td>
			<td>9040-0078</td>
			<td></td>
		</tr>
		<tr>
			<td>Cage Body Eurostandard Type II</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td>267x207x140mm, Floor area 370cm2</td>
		</tr>
		<tr>
			<td>Cell Culture Petri Dish With Lid</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Working Volume: 5mL</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>Microcentrifuge 5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Cuvetta semi-micro L. Form</td>
			<td>Kartell S.p.A.</td>
			<td>01938-00</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Potassium hydrogen phosphate trihydrate</td>
			<td>Carlo erba</td>
			<td>471767</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate anhydrous ACS-for analysis</td>
			<td>Carlo Erba</td>
			<td>480141</td>
			<td>g1000</td>
		</tr>
		<tr>
			<td>Diete Standard Certificate</td>
			<td>Mucedola s.r.l.</td>
			<td>4RF21</td>
			<td>Food pellet for animal</td>
		</tr>
		<tr>
			<td>Dumont Hp Tweezers 5 Stainless Steel</td>
			<td>F.S.T. by DUMONT</td>
			<td>AGT5034</td>
			<td>0,10 x 0,06 mm tip</td>
		</tr>
		<tr>
			<td>Electronic Balance</td>
			<td>Gibertini</td>
			<td>EU-C1200</td>
			<td>Max 1200g, d=0,01g, T=-1200g</td>
		</tr>
		<tr>
			<td>Eppendorf Microcentrifuge tube safe-lock</td>
			<td>Eppendorf</td>
			<td>T3545-1000EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Erythromycin</td>
			<td>Sigma-Aldrich</td>
			<td>E-6376</td>
			<td>25g</td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>F.S.T.</td>
			<td>14084-08</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Filter Top (mini- Isolator), H-Temp with lock clamps</td>
			<td>Tecniplast</td>
			<td>1264C400SUC</td>
			<td></td>
		</tr>
		<tr>
			<td>GC agar base</td>
			<td>OXOID</td>
			<td>CM0367</td>
			<td></td>
		</tr>
		<tr>
			<td>Gillies Forceps 1 x2 teeth</td>
			<td>F.S.T.</td>
			<td>11028-15</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Glicerin RPE</td>
			<td>Carlo Erba</td>
			<td>453752</td>
			<td>1L</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>F.S.T.</td>
			<td>11052-10</td>
			<td>Serrated Tip Width: 0.8mm</td>
		</tr>
		<tr>
			<td>Inner lid</td>
			<td>Tecniplast</td>
			<td>1264C116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron dextran solution</td>
			<td>Sigma-Aldrich</td>
			<td>D8517-25ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbiological Safety Cabinet BH-EN and BHG Class II</td>
			<td>Faster</td>
			<td>BH-EN 2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes 1.5ml&#160;</td>
			<td>BRAND</td>
			<td>PP780751</td>
			<td>screw cap PP, grad</td>
		</tr>
		<tr>
			<td>Mouse Handling Forceps</td>
			<td>F.S.T.</td>
			<td>11035-20</td>
			<td>Serrated rubber; Gripping surface:15 x 20 mm</td>
		</tr>
		<tr>
			<td>Mucotit-F2000</td>
			<td>MERZ</td>
			<td>61846</td>
			<td>2000ml</td>
		</tr>
		<tr>
			<td>Natural Latex Gloves</td>
			<td>Medica</td>
			<td>M101</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Classic C24 Incubator Shaker</td>
			<td>PBI international</td>
			<td>C-24 Classic Benchtop Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri PS Dishes</td>
			<td>VWR</td>
			<td>391-0453</td>
			<td>90X14.2MM</td>
		</tr>
		<tr>
			<td>Pipetman Classic P20</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td>2-20microL</td>
		</tr>
		<tr>
			<td>Pipetman Classic P200</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td>20-200microL</td>
		</tr>
		<tr>
			<td>Pipetman Classim P1000</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td>200-1000microL</td>
		</tr>
		<tr>
			<td>Polyvitox</td>
			<td>OXOID</td>
			<td>SR0090A</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0208</td>
			<td>250g</td>
		</tr>
		<tr>
			<td>Potassium Dihydrogen Phosphate</td>
			<td>J.T. Baker Chemicals B.V.</td>
			<td>0240</td>
			<td>1Kg</td>
		</tr>
		<tr>
			<td>PS Disposible forceps</td>
			<td>VWR</td>
			<td>232-0191</td>
			<td></td>
		</tr>
		<tr>
			<td>Removable Divider</td>
			<td>Tecniplast</td>
			<td>1264C812</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Bottom Polypropylene Tubes</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>5ml</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>MOLEKULA</td>
			<td>41272436</td>
			<td></td>
		</tr>
		<tr>
			<td>SS retainer and Polyester FilterSheet</td>
			<td>Tecniplast</td>
			<td>1264C</td>
			<td></td>
		</tr>
		<tr>
			<td>Standard Pattern Forceps</td>
			<td>F.S.T.</td>
			<td>11000-12</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Stevens Tenotomy Scissors</td>
			<td>F.S.T.</td>
			<td>14066-11</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Surgical Scissor - ToughCut</td>
			<td>F.S.T.</td>
			<td>14130-17</td>
			<td>Stainless</td>
		</tr>
		<tr>
			<td>Touch N Tuff disposible nitrile gloves</td>
			<td>Ansell</td>
			<td>92-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Low Temperature (ULT) Freezer</td>
			<td>Haier</td>
			<td>DW-86L288</td>
			<td>Volume= 288L</td>
		</tr>
		<tr>
			<td>Wagner Scissors</td>
			<td>F.S.T.</td>
			<td>14070-12</td>
			<td>Stainless Steel</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

inducing meningococcal meningitis serogroup c in mice via intracisternal delivery

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. Meningococcus is a transient colonizer of human nasopharynx of approximately 10% of healthy subject. In particular circumstances, it acquires an invasive ability to penetrate the mucosal barrier and invades the bloodstream causing septicaemia. In the latest case, fulminating sepsis could arise even without the consequent development of meningitis. Conversely, bacteria could poorly multiply in the bloodstream, cross the blood brain barrier, reach the central nervous system, leading to fulminant meningitis. The murine models of bacterial meningitis represent a useful tool to investigate the host-pathogen interactions and to analyze the pathogenetic mechanisms responsible for this lethal disease. Although, several experimental model systems have been evaluated over the last decades, none of these were able to reproduce the characteristic pathological events of meningococcal disease. In this experimental protocol, we describe a detailed procedure for the induction of meningococcal meningitis in a mouse model based on the intracisternal inoculation of bacteria. The peculiar signs of human meningitis were recorded in the murine host through the assessment of clinical parameters (e.g., temperature, body weight), evaluation of survival rate, microbiological analysis and histological examination of brain injury. When using intracisternal (i.cist.) inoculum, meningococci complete delivery directly into cisterna magna, leading to a very efficient meningococcal replication in the brain tissue. A 1,000-fold increase of viable count of bacteria is observed in about 18 h. Moreover, meningococci are also found in the spleen, and liver of infected mice, suggesting that the liver may represent a target organ for meningococcal replication.

Survival of mice infected with N. meningitidis wild type and isogenic mutant strains. 
The Neisseria meningitidis strains used in these representative results are the serogroup C reference strain 93/4286 (ET-37) and its isogenic mutant 93/4286&#937;cssA obtained by insertional inactivation of the cssA gene, coding for the UDP-N-acetylglucosamine 2-epimerase, that maps in capsule synthesis locus25. To assess the virulence degree of the c...

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Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

Neisseria meningitidis (meningococcus) is a narrow-host-range microorganism, globally recognized as the leading cause of bacterial meningitis. ...

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Research

JoVE Journal

Immunology and Infection

7.1K Views.  University of Naples Federico II. Here, we describe a method to induce meningococcal meningitis through an intracisternal route of infection in adult mice. We present a step by step protocol of meningococcal infection from the preparation of inoculum to the intracisternal infection; then record the animal survival and evaluate the bacterial loads in murine tissues.

Video: Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the gastrointestinal phase of listeriosis due to the lack of a small animal model that closely mimics human disease. This paper describes a novel mouse model for oral transmission of L. monocytogenes. Using this model, mice fed L. monocytogenes-contaminated bread have a discrete phase of gastrointestinal infection, followed by varying degrees of systemic spread in susceptible (BALB/c/By/J) or resistant (C57BL/6) mouse strains. During the later stages of the infection, dissemination to the gall bladder and brain is observed. The food borne model of listeriosis is highly reproducible, does not require specialized skills, and can be used with a wide variety of bacterial isolates and laboratory mouse strains. As such, it is the ideal model to study both virulence strategies used by L. monocytogenes to promote intestinal colonization, as well as the host response to invasive food borne bacterial infection.

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

This paper describes a novel method for oral infection of mice using Listeria monocytogenes-contaminated food. The protocol can readily be adapted for use with other food borne bacterial pathogens.

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major manifestation of S. suis infections. A suitable infection model is an essential tool to understand the mechanisms of diseases caused by pathogens. Several routes of infection in mice have been developed to study the pathogenesis of S. suis infection. However, the intraperitoneal, intranasal, and intravenous routes of infection are not suitable for studying the roles of S. suis surface components in meningitis directly in the brain, such as the extracellular matrix from biofilms. Although intracisternal inoculation has been used for S. suis infection, the precise injection site has not been described. Here, the intracranial subarachnoidal route of infection was described in a mouse model to investigate the roles of biofilms in S. suis meningitis. S. suis planktonic cells or biofilm state cells were directly injected into the subarachnoid space of mice through the injection site located 3.5 mm rostral from the bregma. Histopathological analysis and increased mRNA expression of TLR2 and cytokines of the brain tissue from mice injected with biofilm state cells clearly indicated that S. suis biofilm plays definitive roles in S. suis meningitis. This route of infection has obvious advantages over other routes of infection, allowing the study of the host-bacterium interaction. Furthermore, it permits the effect of bacterial components on host immune responses directly in the brain to be assessed, and mimics bacterial entrance into the central nervous system. This route of infection can be extended for investigating the mechanisms of meningitis caused by other bacteria. In addition, it can also be used to test the efficacy of drugs against bacterial meningitis.

Intracranial Subarachnoidal Route of Infection for Investigating Roles of Streptococcus suis Biofilms in Meningitis in a Mouse Infection Model

Here, we describe the intracranial subarachnoidal route of infection in mice to study roles of biofilms in Streptococcus suis meningitis. This infection model is also suitable for studying the pathogenesis of other bacterial meningitis and the efficacy of new drugs against bacterial meningitis.

Streptococcus suis is not only a major bacterial pathogen of pigs worldwide but also an emerging zoonotic agent. In humans and pigs, meningitis is a major ...

Quantitative PCR of T7 Bacteriophage from Biopanning

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a fluorescence-based approach to quantify DNA, and here, it is adapted to quantify phage genomes as a proxy for phage particles. In this protocol, a facile phage DNA preparation method is described using high-temperature heating without additional DNA purification. The method only needs small volumes of heat-treated phages and small volumes of the qPCR reaction. qPCR is high-throughput and fast, able to process and obtain data from a 96-well plate of reactions in 2&#8211;4 h. Compared to other phage enumeration approaches, qPCR is more time-efficient. Here, qPCR is used to enumerate T7 phages identified from biopanning against in vitro cystic fibrosis-like mucus model. The qPCR method can be extended to quantify T7 phages from other experiments, including other types of biopanning (e.g.,&#160;immobilized protein binding, in vivo phage screening) and other sources (e.g., water systems or body fluids). In summary, this protocol can be modified to quantify any DNA-encapsulated viruses.

A reproducible, accurate, and time efficient quantitative PCR (qPCR) method to enumerate T7 bacteriophage is described here. The protocol clearly describes phage genomic DNA preparation, PCR reaction preparation, qPCR cycling conditions, and qPCR data analysis.

This protocol describes the use of quantitative PCR (qPCR) to enumerate T7 phages from phage selection experiments (i.e., &#34;biopanning&#34;). qPCR is a ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...