Immunolabeling Of Yersinia pestis To Determine Early Colonization Of The Flea Proventriculus Using SEM. C. D. Pauling1, D. Anderson1

1Department of Veterinary Pathobiology, University of Missouri, Columbia, Missouri 65211 USA Immunolabeling of Yersinia pestis was utilized to positively identify the localization of bacteria within the flea at day 3 post-infection with Scanning Electron Microscopy (SEM). Current research implicates two transmission mechanisms of Y. pestis by the flea, one called Early-Phase Transmission ,EPT, which occurs days 1-4 post-infection, and a biofilm-mediated transmission that can occur as early as 7 days post-infection but typically occurs days 14-28 post-infection [1,2]. Research in flea transmission has implicated genes required for colonization and biofilm formation in the flea that include the ymt gene which allows colonization of the midgut and the hms gene responsible for production of exopolysaccharides required for biofilm formation [3,4]. However, these genes have been shown to not be relevant for EPT, leaving the mechanisms behind this transmission model unknown [5,6]. The goal of this research was to determine if Y. pestis can colonize the proventriculus during the time post-infection that correlates to EPT. SEM provides a sensitive method to view bacterial colonization within the proventriculus. Previous research in our lab utilizing SEM indicated a possible biofilm associated structure on the proventriculus early, however; we needed to definitively identify the bacteria associated with this structure as Y. pestis. This is the first step in our research which will focus on factors that allow Y. pestis to colonize the chitinous proventriculus. Additionally, we are investigating the kinetics of Y. pestis acquisition and colonization within the flea to help determine mechanisms behind EPT. The structure previously identified on the proventriculus is hypothesized to play a major role in both EPT and biofilm-mediated transmission, therefore, the composition and dynamics are vital to understand. Initial immunolabeling procedures attempted to label bacteria with quantum dots so that the samples may be analyzed by both SEM and fluorescent confocal microscopy. Quantum dots are a nanoparticle made of a semiconducting material such as cadmium and zinc. Due to the small size of the quantum dots, which range from 2-10 nm, we were unable to identify quantum dots utilizing SEM or detect the materials associated with the quantum dots. Therefore, we altered the immunolabeling methods to incorporate gold nanoparticles. A primary anti-Yersinia antibody was biotinylated and added to a bacterial overnight and incubated at room temperature. A secondary biotinylated anti-IgG mouse antibody was incubated with streptavidin coated 20nm gold nanoparticles at room temperature. The bacterial overnight was then spun down, supernatant was removed, and PBS was added. The primary plus bacteria and the secondary plus gold nanoparticles were then incubated together at room temperature for 45 minutes. The mixture was spun down, supernatant was removed and then the bacteria was added to a bloodmeal fed to fleas using an artificial feeder. The methods successfully immunolabeled Y. pestis as represented in the figures below. Additionally, at day 3 post-infection, immunolabeled bacteria was detected in the proventricular mass that was present. Only a small portion is detected as bacteria replicates within the flea and appears to be

quenched in the forming structure. The detection of Y.pestis in the proventriculus during early infection indicates colonization of the proventriculus eluding to additional colonization mechanisms occurring to allow for adherence. Future studies will involve methods to investigate gene regulation of Y. pestis, biofilm production and the functional role of the microbiome in acquisition and colonization [7]. References: [1] Eisen, R.J., et al. 2006. Early-phase transmission of Yersinia pestis by unblocked fleas as a mechanism explaining rapidly spreading plague epizootics. National Academy of Science. 103(42): 15380-15385. [2] Lorange, E.A., et al. 2005. Poor vector competence of fleas and the evolution of hypervirulence in Yersina pestis. The Journal of Infectious Diseases. 191: 1907-1912. [3] Vadyvaloo, V., et al. 2010. Transit through the flea vector induces a pretransmission innate immunity resistance phenotype in Yersinia pestis. PLOS Pathogens. 6(2): e1000783. [4] Zhou, D. and R. Yang. 2011. Formation and regulation of Yersinia biofilms. Protein and Cell. 2(3): 173-179. DOI 10.1007/s13238-011-1024-3. [5] Vetter, S.M., et al. 2010. Biofilm formation is not required for early-phase transmission of Yersinia pestis. Microbiology 156:2216-2225. [6] Johnson, T.L. et al. 2014. Yersinia murine toxin is not required for early-phase transmission of Yersinia pestis by Oropsylla montana (Siphonaptera: Ceratophyllidae) or Xenopsylla cheopis (Siphonaptera: Pulicidae). Microbiology 160: 2517-2525. [7] Acknowledgments: David Stalla and Deana Grace at University of Missouri EM Core. Funding provided by the University of Missouri EM Core.

Fig. 1. Immunolabeled Yersinia pestis with gold nanoparticles.

Fig. 2. Immunolabeled Yersinia pestis in the flea proventriculus on Day 3 post-infection. Arrows indicated immunolabeled Yersinia pestis.