TITLE: Infection of mice with Citrobacter rodentium ICC180 by natural transmission
AUTHORS: Hannah Read, Priyali Patel (University of Auckland), Siouxsie Wiles

[DESCRIPTION]
Citrobacter rodentium is a Gram-negative bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. We routinely use a bioluminescent derivative of C. rodentium called ICC180 which allows us to monitor infection dynamics non-invasively using biophotonic imaging.

Experimental infection is typically initiated via oral gavage but we have previously shown that C. rodentium rapidly spreads between infected and uninfected animals and that bacteria shed from infected mice are 1000 times more infectious than laboratory-grown bacteria with a different tissue tropism.1,2 In this protocol, we describe the different methods for establishing an infection using natural transmission.  

1. Wiles, S., Dougan, G., & Frankel, G. (2005). Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract. Cellular microbiology, 7(8), 1163–1172. https://doi.org/10.1111/j.1462-5822.2005.00544.x
2. Bishop, A. L., Wiles, S., Dougan, G., & Frankel, G. (2007). Cell attachment properties and infectivity of host-adapted and environmentally adapted Citrobacter rodentium.Microbes and infection,9(11), 1316–1324. https://doi.org/10.1016/j.micinf.2007.06.006

[BEFORE_START]
Prior to oral gavage, ensure animals have been weighed and marked in some way so that you can identify individual animals. We weigh animals by placing them in a 1ml pipette tip box (with the insert removed) placed on a set of scales.  We use a marker pen to mark each animal's tail. For example, if there are 5 animals in a cage, we give each animal 1-5 lines across their tails.  In our experience, a black marker pen stays visible the longest.   

[GUIDELINES]
Experiments involving animals and pathogenic bacteria require ethical and biological safety approval. When planning experiments involving animals, consult the PREPARE and ARRIVE guidelines.

[STEPS]
SECTION: Preparation of Citrobacter rodentium ICC180
1. At least two days before needed, revive bacteria from frozen stocks stored at –70-80°C. Plate onto LB-Lennox media. At this stage, you can grow them with or without kanamycin 50 ug/mL . Incubate  1440 min  at 37 °C 

SECTION: Preparation of Citrobacter rodentium ICC180
2. The day before needed, inoculate 10 mL LB-Lennox (LB) media supplemented with kanamycin in a 50 mLtube. We generally inoculate using several colonies to provide a more heterogeneous culture for infection. Incubate 1440 min  at 37 °C with shaking at  .

SECTION: Preparation of Citrobacter rodentium ICC180
3. On the day of infection, centrifuge the culture at 4500 rpm for 5 min and resuspend in 1 mL PBS to give a 10x concentrated inoculum.

SECTION: Preparation of Citrobacter rodentium ICC180
4. To retrospectively calculate the number of bacteria in the inoculum, prepare a 10-fold dilution series of the inoculum in PBS and incubate 3 25 µL drops of each dilution onto LB plates (with or without kanamycin). Incubate overnight at 37 °C and count the colonies. 

SECTION: Oral gavage of seed mice
5. [Optional] Animals can be lightly anaesthetised using gaseous isoflurane to aid gavage. To do this, place mice into the anaesthetic induction chamber and induce anaesthesia using a flow rate of 1 L/min oxygen combined with 5% isoflurane. Animals are sufficiently anaesthetised once the animals have lost their righting reflex. 

It is important that animals are not too deeply anaesthetised as their vital functions can be compromised. The respiratory rate of a normal undisturbed mouse is approximately 180 breaths per minute. A slow rate drop of 50% is acceptable during anaesthesia. Breathing should be steady. If the animals’ breathing becomes ‘‘jerky’’, too much anaesthetic is being applied and this will be fatal if maintained for long periods of time. If an animal appears too deeply anaesthetised, immediately turn off the anaesthetic and administer supplemental oxygen. 
SECTION: Oral gavage of seed mice
7. Using the feeding needle, orally gavage each animal with 200 µL of concentrated inoculum. 

This video is a good resource for people who are new to the technique: https://researchanimaltraining.com/articles/oral-gavage-in-the-mouse/. 

To minimise the risk of oesophageal trauma and incorrect dosing, it is crucial that the operator is skilled both in the technique and the restraint method used. Inadvertent dosing into the lung may occur, and this usually results in the animal showing immediate signs of respiratory distress. If this is observed, then the animal should be humanely killed using an approved method.

SECTION: Oral gavage of seed mice
6. Transfer the inoculum to a 1 mL syringe and attach a feeding needle. 

SECTION: Oral gavage of seed mice
8. After dosing, return animals to their cage and observe. If done correctly, the animals should resume normal activity within minutes.  
SECTION: Oral gavage of seed mice
9. Animals should be routinely monitored by measuring their weight, behaviour, and condition. The GRIMACE scale is ideal. The original study that developed the scale is online here and an explanatory poster and other resources are available here. 

Depending on the size of the dose, some animals may not eat for a short period and so may experience some weight loss in the first 24 hours after gavage. If they are active and alert and their fur remains smooth and glossy, this is usually no cause for concern.
SECTION: Natural transmission of C. rodentium from seed to naive mice
10. At the peak of infection (usually between days 6-10 post-gavage) move one or more infected mice to a clean cage.  
SECTION: Natural transmission of C. rodentium from the environment
13. [Optional] Instead of cohousing naive and infected mice together, infection can be initiated from a contaminated environment.  At the peak of infection (usually between days 6-10 post-gavage), transfer infected animals to a clean cage. 
SECTION: Natural transmission of C. rodentium from the environment
14. After 18-48 hours, remove infected animals from the cage and remove some stool from the cage into a preweighed microcentrifuge tube. This will enable you to retrospectively calculate the level of environmental C. rodentium contamination. 
SECTION: Natural transmission of C. rodentium from the environment
15. Add naive animals to the dirty cage. C. rodentium will transmit via coprophagia and generally occurs within 48 hours. 
SECTION: Natural transmission of C. rodentium from seed to naive mice
11. Add naive animals to the clean cage alongside the infected animals. C. rodentium will transmit via coprophagia and grooming. Transmission usually occurs within 48 hours. 
SECTION: Natural transmission of C. rodentium from seed to naive mice
12. All animals should be routinely monitored by measuring their weight, behaviour, and condition. The GRIMACE scale is ideal. The original study that developed the scale is online here and an explanatory poster and other resources are available here. 
SECTION: Natural transmission of C. rodentium from the environment
16. To retrospectively calculate the level of environmental C. rodentium contamination, add PBS to a final concentration of 0.1 g of stool per 1 mL  of PBS. Vortex briefly, then prepare a 10-fold dilution series in PBS and incubate 3 25 µL drops of each dilution onto LB plates supplemented with 50 ug/mL kanamycin. Incubate overnight at 37 °C and count the colonies. 

SECTION: Establishing a transmission chain
17. [Optional] To establish a defined transmission chain, co-house one infected mouse (usually around the peak of infection) and one naive mouse together in a clean cage. C. rodentium should transmit via coprophagia and grooming within 48 hours. 
SECTION: Establishing a transmission chain
19. If transmission fails to occur,  re-established the transmission chain using an inoculum derived from the stool homogenate frozen in glycerol. Use a sample of the stool homogenate to inoculate  10 mL LB-Lennox (LB) media supplemented with kanamycin in a 50 mLtube. Incubate 1440 min  at 37 °C with shaking at   and oral gavage as previously described.

SECTION: Establishing a transmission chain
18. Around the peak of infection, collect stool samples from infected mice to monitor infection levels and reinitiate the transmission chain should natural transmission fail.
SECTION: Establishing a transmission chain
18.1. Label and weigh individual microfuge tubes. Write the weight on the side of the tube.
Remove inserts from pipette tip boxes
SECTION: Establishing a transmission chain
18.2. Place the infected mouse in a clean pipette tip box. Once the mouse has produced 1-3 individual stools, transfer these to a sterile labelled microfuge tube of known weight using a pair of clean tweezers. 


Depending on the mouse strain, stools can become very loose and much more difficult to pick up using tweezers. In these instances, try using a small laboratory spatula instead. We've also observed mice placing their stools up on the walls of the tip box like they are moving them out of the way or making an offering.   
SECTION: Establishing a transmission chain
18.3. Transfer the mouse back to its home cage and clean the pipette tip box.
SECTION: Establishing a transmission chain
18.4. Weigh the tube again to find the weight of the stool.
SECTION: Establishing a transmission chain
18.5. Add PBS to a final concentration of 0.1 g  of stool per 1 mL of PBS. Homogenise using a vortex.

SECTION: Establishing a transmission chain
18.6. To retrospectively determine the number of bacteria present, prepare a 10-fold dilution series in PBS and incubate 3 25 µL drops of each dilution onto LB plates supplemented with 50 ug/mL kanamycin. Incubate overnight at 37 °C and count the colonies. 
SECTION: Establishing a transmission chain
18.7. To the remainder of the stool homogenate, add an equal volume of 50 % (v/v) glycerol to give a final concentration of 25 % (v/v) and store at -70 °C or below.