Inducing Meningococcal Meningitis Serogroup C in Mice via Intracisternal Delivery

Summary

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.

Abstract

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.

Introduction

Neisseria meningitidis is a Gram negative β-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 behavior

Protocol

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

Results

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 93/4286ΩcssA 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 cssA

Discussion

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,8 Skirted Cryovial With external thread	Starlab	E3090-6222
50 mL Polypropylene Conical Tube	Falcon	352070	30 mm x 115 mm
Adson Forceps	F.S.T.	11006-12	Stainless Steel
Alarm-Thermometer	TESTO	9000530
BactoTM Proteose Peptone	BD	211693
BD Micro Fine syringe	BD	320837	U-100 Insulin
BD Plastipak syringe 1 mL 25 G 5/8 inch	BD	300014	05 mm x 16 mm
BD Plastipak syringe 5 mL	BD	308062	07 mm x 30 mm
BIOHAZARD AURA B VERTICAL LAMINAR FLOW CABINET	Bio Air s.c.r.l.	Aura B3
BioPhotometer	Eppendorf	Model #6131
Bottle D	Tecniplast	D	Graduated up to: 400 mL, Total Volume 450 mL, 72 mm x 72 mm x 122 mm
C150 CO2 Incubator	Binder	9040-0078
Cage Body Eurostandard Type II	Tecniplast	1264C	267 x 207 x 140 mm3, Floor area 370 cm2
Cell Culture Petri Dish With Lid	Thermo Scientific	150288	Working Volume: 5 mL
Centrifuge	Eppendorf	Microcentrifuge 5415R
Cuvetta semi-micro L. Form	Kartell S.p.A.	01938-00
di-Potassium hydrogen phosphate trihydrate	Carlo erba	471767
di-Sodium hydrogen phosphate anhydrous ACS-for analysis	Carlo Erba	480141	g1000
Diete Standard Certificate	Mucedola s.r.l.	4RF21	Food pellet for animal
Dumont Hp Tweezers 5 Stainless Steel	F.S.T. by DUMONT	AGT5034	0.10 x 0.06 mm2 tip
Electronic Balance	Gibertini	EU-C1200	Max 1200 g, d = 0.01 g, T = -1200 g
Eppendorf Microcentrifuge tube safe-lock	Eppendorf	T3545-1000EA
Erythromycin	Sigma-Aldrich	E-6376	25 g
Extra Fine Bonn Scissors	F.S.T.	14084-08	Stainless Steel
Filter Top (mini- Isolator), H-Temp with lock clamps	Tecniplast	1264C400SUC
GC agar base	OXOID	CM0367
Gillies Forceps 1 x2 teeth	F.S.T.	11028-15	Stainless Steel
Glicerin RPE	Carlo Erba	453752	1 L
Graefe Forceps	F.S.T.	11052-10	Serrated Tip Width: 0.8 mm
Inner lid	Tecniplast	1264C116
Iron dextran solution	Sigma-Aldrich	D8517-25ML
Ketamine	Intervet
Microbiological Safety Cabinet BH-EN and BHG Class II	Faster	BH-EN 2004
Microcentrifuge tubes 1.5 mL	BRAND	PP780751	screw cap PP, grad
Mouse Handling Forceps	F.S.T.	11035-20	Serrated rubber; Gripping surface: 15 mm x 20 mm
Mucotit-F2000	MERZ	61846	2000 mL
Natural Latex Gloves	Medica	M101
New Brunswick Classic C24 Incubator Shaker	PBI international	C-24 Classic Benchtop Incubator Shaker
Petri PS Dishes	VWR	391-0453	90 x 14.2 mm2
Pipetman Classic P20	Gilson	F123600	2-20 μL
Pipetman Classic P200	Gilson	F123601	20-200 μLL
Pipetman Classim P1000	Gilson	F123602	200-1,000 μL
Polyvitox	OXOID	SR0090A
Potassium Chloride	J.T. Baker Chemicals B.V.	0208	250 g
Potassium Dihydrogen Phosphate	J.T. Baker Chemicals B.V.	0240	1 kg
PS Disposible forceps	VWR	232-0191
Removable Divider	Tecniplast	1264C812
Round-Bottom Polypropylene Tubes	Falcon	352063	5 mL
Sodium Chloride	MOLEKULA	41272436
SS retainer and Polyester FilterSheet	Tecniplast	1264C
Standard Pattern Forceps	F.S.T.	11000-12	Stainless
Stevens Tenotomy Scissors	F.S.T.	14066-11	Stainless Steel
Surgical Scissor - ToughCut	F.S.T.	14130-17	Stainless
Touch N Tuff disposible nitrile gloves	Ansell	92-500
Ultra Low Temperature (ULT) Freezer	Haier	DW-86L288	Volume = 288 L
Wagner Scissors	F.S.T.	14070-12	Stainless Steel
Xylazine	Intervet