Infant Rabbit Colonization Competition Assays    

Materials and Reagents

1. New Zealand white infant rabbit (2-5 days old) (Millbrook farm)
   
2. Lactating doe (Millbrook farm)
   
3. Vibrio cholerae
   Note: Alternatively, any enteric pathogenic bacteria that can successfully colonize the infant rabbit gut system can be used in this protocol.
   
4. Zantac (GlaxoSmithKline, NDC: 0173-0363-01 )
   
5. 40 mEq/20 ml potassium chloride (KCl) (Hospira, NDC: 0409-6653-05 )
   
6. Isoflurane (USP) (Piramal Enterprises, NDC: 66794-013-25 )
   
7. Phosphate buffered saline (PBS) (Lonza, catalog number: 51225 )
   
8. X-gal and/or antibiotics (depend on the selective marker of the experimental strain)
   
9. 70% ethanol
   
10. 2.5% sodium bicarbonate buffer (pH 8.0) (see Recipes)
    
11. Luria-Bertani broth (LB) medium and LB agar solid medium (see Recipes)
    

Equipment

1. BL2 animal facility
   
2. Fume hood
   
3. 37 °C incubator or warm room
   
4. 1 ml sterile syringe (BD, catalog number: 305553 )
   
5. 3 ml sterile syringe (BD, catalog number: DG508504 )
   
6. 26 G 5/8 needle (BD, catalog number: 305115 )
   
7. Surgical scissors and tweezers (Fine Science Tools)
   
8. Centrifuge (Eppendorf, model: 5424 )
   
9. 1.5 ml sterile microcentrifuge tube
   
10. 96 wells tissue culture plate (BD, catalog number: 353072 )
    
11. Size 5 French catheter
    
12. Silk ligature
    
13. Shaker
    
14. Spectrophotometer
    
15. Mini-bead beater-16 (Bio Spec Products, catalog number: 607 )
    

Procedure

1. Check the litters on the day when they arrive: Litters of 2-5 day old New Zealand white rabbits and the lactating doe are obtained from a commercial vendor and housed together for the duration of the experiments. A minimum of six rabbits need to be assayed for each strain to be evaluated.
   
2. Inocula preparation: A single colony from a freshly streaked plate of each mutant (lacZ+) and wild type (lacZ-) is grown in LB containing the selective antibiotic of the experimental strains for approximate 5 to 6 h at 37 °C with shaking. The bacterial cells are pelleted (9,000 rpm for 5 min), the supernatant is discarded and the pellet is resuspended in sterile sodium bicarbonate buffer. The concentration of bacteria is adjusted to about 2 x 10⁹ cfu/ml. Wild type and mutant solutions are then mixed together (1:1).
   
3. Zantac treatment (3 h prior to inoculation): Infant rabbits are weighed and administered Zantac by intraperitoneal (i.p) injection (2 mg per kg body weight) using a 26 G needle and 1 ml syringe. (This dose of anti-acid treatment has been found to transiently alter the pH of the stomach without causing ill-effects in the infant rabbits.).  
   
4. 3 h post-Zantac treatment: Infant rabbits are oro-gastrically inoculated with 0.5 ml of bacteria using a size 5 French catheter with flexible tip. The suggested dose is 1 x 10⁹ cfu/ml per 90 g of rabbit body weight.
   
5. Input ratio determination: Inocula are serial diluted in 1x PBS and plated on LB agar containing X-gal. CFU of white and blue colonies are counted after overnight incubation at 37 °C.
   
6. Clean the catheter: Wash the catheter with 70% ethanol, H₂O and 1x PBS sequentially.
   
7. Monitor rabbits: Infant rabbits are monitored for diarrhea and signs of illness (e.g. dehydration, low body temperature, scruffy fur, lethargy and decreased muscle tone) and routinely euthanized within few days.
   
8. Euthanasia: Infant rabbits are anesthetized by inhalation of isoflurane and euthanized by intracardiac injection of KCl (2 mEq/ml, 3 ml).
   
9. Necropsy: The entire intestinal tract from the duodenum to the rectum is removed.
   1. Cecal fluid: Cecal fluid (1-2 ml per rabbit) is collected by first isolating the cecum from the rest of the intestine with silk ligatures; then, the cecal contents are collected by snipping the end of the cecum and allowing the contents to drain under gravity into a collection tube. 1 ml cecal fluid of each rabbit is used for the CFU counting.
      
   2. Distal small intestine and colon: A 1 cm sample is cut and removed, and then homogenized (e.g. bead beater or slides scissoring) in 1 ml of 1x PBS.
10. Output ratio determination: Serial dilutions of each sample are plated in LB solid media with antibiotic and X-gal and incubated overnight at 37 °C to enumerate the output ratio of the wild type and mutant strain.
    
11. Competitive Index (C.I) counting: The competitive index for each mutant is defined as the input ratio of mutant/WT strain divided by the output ratio of mutant/WT strain. (Figure 1)
    

Representative data

1. Example of infant rabbit competitive index counting
   
   
   Figure 1. Infant rabbit competition assays of intestinal colonization related mutants of Vibrio cholerae C6706 strain. In vivo competition experiments were performed comparing the colonization related mutants to parental strain C6706 lacZ- (WT). Left, negative control mutant C6706 lacZ+; Middle left, severe colonization defective mutantΔtcpA; Middle right, intermediate defect mutant ΔdncV and Right, hyper-colonization mutant ΔvspR. Statistical significance was determined by t test relative to the C.I. determined for a competition between C6706 lacZ+ versus WT with **** indicating a P value < 0.0001, *** P< 0.001. Mean with SEM was shown.
   

Notes

1. Ethics statement
   All animal experiments were performed with protocols approved by the Harvard Medical School Office for Research Protection Standing Committee on Animals in accordance to NIH guidelines. The Harvard Medical School animal management program is also accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). The institution also accepts as mandatory the PHS Policy on Humane Care and Use of Laboratory Animals by Awardee Institutions and NIH Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training. The office of Laboratory Animal Welfare (OLAW) has the approved Assurance of Compliance (A3431-01) on file.
   

Recipes

1. 2.5% sodium bicarbonate buffer (pH 8.0) (100 ml)
   Mix 2.5 g Sodium Bicarbonate with 80 ml dH₂O
   pH to 8.0 with NaOH
   Add dH₂O to 100 ml
   Filter sterilize (0.2 μm)
   Stored at room temperature
   
2. Luria-Bertani broth (LB) medium (1 L)
   Mix of 10 g tryptone, 10 g NaCl and 5 g yeast extract
   Add dH₂O to 1 L
   For LB agar add agar to a final concentration of 1.5%
   Heat the mixture to boiling to dissolve agar and sterilize by autoclaving at 15 psi, from 121-124 °C for 15 min
   Stored at room temperature
   

Acknowledgments

This protocol was first described and used in our previous Vibrio cholerae intestinal colonization study (Fu et al., 2013), and was adapted from a precious infant rabbit model, which was described by Ritchie et al. (2010). We thank Dr. Waldor and Dr. Ritchie for helpful suggestions and comments. This work was funded by grants AI-018045 and AI-26289 to J.J.M. from the National Institute of Allergy and Infectious Disease.

References

1. Fu, Y., Waldor, M. K. and Mekalanos, J. J. (2013). Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14(6): 652-663.
   
2. Ritchie, J. M., Rui, H., Bronson, R. T. and Waldor, M. K. (2010). Back to the future: studying cholera pathogenesis using infant rabbits. MBio 1(1).