|GRASSMANN, ANDRE - University Of Connecticut|
|MCBRIDE, ALAN - Universidade Federal De Pelotas|
|CAIMANO, MELISSA - University Of Connecticut|
Submitted to: Bio-protocol
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/17/2015
Publication Date: 7/20/2015
Citation: Grassmann, A.A., Mcbride, A.J., Nally, J.E., Caimano, M.J. 2015. Generation of mammalian host-adapted Leptospira interrogans by cultivation in peritoneal dialysis membrane chamber implantation in rats. Bio-protocol. 5(14). pii: e1536.
Interpretive Summary: Pathogenic leptospires cause leptospirosis, a zoonotic disease that is disseminated via urine of persistently infected mammalian host species. Excreted leptospires continue to survive in a suitable moist environment and infect new hosts by penetrating mucosal surfaces or breaches of the skin. Thus, leptospires have an innate ability to adapt to, and survive in, a range of different environmental conditions. To further understand how leptospires adapt to mammalian host signals encountered during infection, a novel protocol has been developed to cultivate pathogenic species of Leptospira in dialysis membrane chambers which are subsequently implanted into the peritoneal cavity of rats. In this way, large numbers of leptospires adapted to grow in response to mammalian host signals can be generated and used as a source of “host-adapted” leptospires for experimental research purposes. This manuscript provides a step-by-step protocol to cultivate “host-adapted” leptospires within dialysis membrane chambers in the peritoneal cavity of rats.
Technical Abstract: Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti, Nally et al. 2003, Ko, Goarant et al. 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazio, Silva et al. 2008). Infected hosts shed large number of spirochetes in their urine and the leptospires can survive in different environmental conditions before transmission to another host. Differential gene expression by Leptospira spp. permits adaption to these new conditions. Here we describe a protocol for the cultivation of Leptospira interrogans within Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimano, Sivasankaran et al. 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete, Borrelia burgdorferi (Akins, Bourell et al. 1998, Caimano 2005). The small pore size (8000 MWCO) of the dialysis membrane tubing used for this procedure permits access to host nutrients but excludes host antibodies and immune effector cells. Given the physiological and environmental similarities between DMCs and the proximal convoluted renal tubule, we reasoned that the DMC model would be suitable for studying in vivo gene expression by L. interrogans. In a 20 to 30 min procedure, DMCs containing virulent leptospires are surgically-implanted into the rat peritoneal cavity. Nine to 11 days post-implantation, DMCs are explanted and organisms recovered. Typically, a single DMC yields ~109 mammalian host-adapted leptospires (Caimano, Sivasankaran et al. 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenic L. interrogans undergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants.