幼兔肠道中不同菌株的定植竞争分析

Yang  Fu; John J.  Mekalanos

doi:10.21769/BioProtoc.1147

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Peer-reviewed

Infant Rabbit Colonization Competition Assays

幼兔肠道中不同菌株的定植竞争分析

Yang Fu

JM John J. Mekalanos email

发布: 2014年06月05日第4卷第11期 DOI: 10.21769/BioProtoc.1147 浏览次数: 10338

评审: Anonymous reviewer(s)

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参见作者原研究论文

The authors used this protocol in:

Cover of Cell Host & Microbe, featuring study using the protocol.

Dec 2013

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Abstract

Enteric pathogenic bacteria such as Vibrio cholerae and enteropathogenic Escherichia coli (E. coli) cause life-threatening diarrheal diseases that have afflicted humans for centuries. Understanding the effectors required for intestinal colonization is very important to research on bacteria pathogenesis, and is also important to testing new therapeutics and development of the novel vaccines. Here, we describe the Infant Rabbit Colonization Competition Assay, a variant method of the powerful, nonsurgical animal model reported by Ritchie et al. (2010). In our modified assay, wild type and mutant strains are mixed together and inoculated into 2-day-old New Zealand white rabbits. The competitive index for each mutant measures the colonization capacity of the mutant relative to its wild type parental strain in the gastrointestinal tract. Compared to the traditional Sucking Mice model, the clinical and histologic signs of Vibrio cholerae (V. cholerae)-induced disease of infant rabbits more closely resemble human cholera. The larger input bacteria amount of this model also facilitates high-throughput screens, such as Tn-Seq technology (Fu et al., 2013).

Keywords: Vibrio Cholerae (霍乱弧菌)

Enteric Bacteria (肠道细菌)

Animal Model (动物模型)

Colonization Factor (定植因子)

Competetion Assay (竞争分析)

Materials and Reagents

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