title: Generation of Mammalian Host-adaptedLeptospira interrogansby Cultivation in Peritoneal Dialysis Membrane Chamber Implantation in Rats.
abstract: Leptospira interroganscan infect a myriad of mammalian hosts, including humans (Bhartiet al., 2003; Koet al., 2009). Following acquisition by a suitable host, leptospires disseminateviathe bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize the proximal convoluted renal tubules (Athanazioet 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 byLeptospiraspp. permits adaption to these new conditions. Here we describe a protocol for the cultivation ofLeptospira interroganswithin Dialysis Membrane Chambers (DMCs) implanted into the peritoneal cavities of Sprague-Dawley rats (Caimanoet al., 2014). This technique was originally developed to study mammalian adaption by the Lyme disease spirochete,Borrelia burgdorferi(Akinset al., 1998; Caimano, 2005). The small pore size (8,000 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 studyingin vivogene expression byL. 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 ~109mammalian host-adapted leptospires (Caimanoet al., 2014). In addition to providing a facile system for studying the transcriptional and physiologic changes pathogenicL. interrogansundergo within the mammal, the DMC model also provides a rationale basis for selecting new targets for mutagenesis and the identification of novel virulence determinants. Caution: Leptospira interrogans is a BSL-2 level pathogen and known to be excreted in the urine of infected animals. Animals should be handled and disposed of using recommended Animal Biosafety Levels (ABSL) for infectious agents using vertebrate animal guidelines. Note: All protocols using live animals must conform to governmental regulations regarding the care and use of laboratory animals. The success of this protocol is dependent on the proper use of aseptic techniques during all stages of both dialysis membrane chamber preparation and animal surgery.
authors.affiliation: Unidade de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil ; Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: André Alex
authors.initials: AA
authors.lastname: Grassmann
authors.affiliation: Unidade de Biotecnologia, Centro de Desenvolvimento Tecnológico, Universidade Federal de Pelotas, Pelotas, Rio Grande do Sul, Brasil.
authors.forename: Alan John Alexander
authors.initials: AJ
authors.lastname: McBride
authors.affiliation: Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, Iowa.
authors.forename: Jarlath E
authors.initials: JE
authors.lastname: Nally
authors.affiliation: Department of Medicine, University of Connecticut Health Center, Farmington, Connecticut, USA ; Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut, USA ; Department of Molecular Biology & Biophysics, University of Connecticut Health Center, Farmington, Connecticut, USA.
authors.forename: Melissa J
authors.initials: MJ
authors.lastname: Caimano