1. The prevalence of co-infections among Ixodes scapularis harvested from freshly slain deer Paul N. Williams, Dept. of Biology, York College, York, PA 17405 Abstract The white-tailed deer is recognized as a reproductive-stage host of the deer tick Ixodes scapularis, a known vector for several pathogenic organisms. One of these organisms is Borrelia burgdorferi, the causative agent of Lyme disease, the most commonly reported vector-borne illness in the United States. Additionally, Ixodes is a known vector for Babesia microti, Bartonella henselae, and Anaplasma phagocytophila, the causative agents of babesiosis, cat-scratch disease, and human granulocytic ehrlichiosis respectively. The presence and transmission of co-infections of these organisms can complicate diagnosis and result in a more severe clinical progression of disease. The objective of this study was the determination of the rates of co-infection of the above organisms in Ixodes found on deer that had been recently slain by hunters. Ticks were harvested over a two day period and PCR was performed to determine the presence of the respective organisms. Additionally, PCR detection of Ixodes was run as an internal control. To date, of 20 examined ticks, one is suspected to have been a vector for both Borrelia and Babesia, while another has been confirmed as a vector for Anaplasma phagocytohpila. Introduction Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most commonly reported vector borne illness in the United States (CDC 2002). Its primary invertebrate vector is the deer tick Ixodes scapularis. Complicating both the diagnosis and prognosis of Lyme disease is the possibility of co-infections with other organisms that utilize Ixodes as a vector. Among the most prevalent of these organisms are Anaplasma phagocytophila, the causative agent of Human Granulocytic Ehrlichiosis, Babesia microti, the organism responsible for babesiosis, and Bartonella henselae, which causes the illness often referred to as “cat- scratch disease.” In addition to sharing Ixodes as a vector, each of these pathogens can potentially take advantage of the immunocompromise that accompanies Lyme disease (Adelson et al. 2004). Objective: To determine the prevalence of co-infections in ticks found on locally slain deer. A C B D A Harvest ticks from slain deer Extracted DNA using Dneasy kit Performed PCR using primers from literature Performed electrophoresis using 8% agarose gel Gel analysis using AlphaEase software Methods The author would like to thank Carolyn Mathur, PhD and Richard Daly, MD, for their assistance in the design of this project, the good people of the Windsor Meat Market for the use of their facilities, Jeffrey Thompson, PhD and Ronald Kaltreider, PhD, for their assistance in the molecular biology bits, and Karl Kleiner, PhD, for his constructive criticism and general oversight of the project. Acknowledgments A C D E Figure 1. Micrographs of A) Borrelia burgdorferi, B) Babesia microti, C) Bartonella henselae, and D) Anaplasma phagocytophila. Ladder CD 215 bp 238 bp 275 bp 185 bp 546 bp Figure 2. Electrophoresis of PCR product from DNA extracted from A) Ixodes scapularis, B) Borrelia burgdorferi, C) Babesia microti, D) Bartonella henselae, and E) Anaplasma phagocytophila. Arrows indicate expected base pair size for each sample. In each gel, lanes 2-10 from the top wells and lanes 2- 11 from the bottom wells represent DNA extracted from individual ticks. Results Electrophoresis failed to confirm the identity of Ixodes in many of the samples There is the possibility of co-infection with Borrelia and Babesia in one of the ticks sampled The presence of Anaplasma phagocytophila was confirmed in a separate deer tick Discussion The absence of expected bands in the Ixodes gel suggests that either DNA extraction or PCR was not entirely effective. However, discernible bands for both Borrelia and Babesia are present in a single tick sample, suggesting the possibility of co- infection. Additionally, nested PCR for Anaplasma revealed an appropriately placed band for one of the ticks, which is strongly indicative of infection. DNA sequencing will be used to confirm. A comparatively small sample size and the possibility of inefficient extraction may have contributed to less- than expected rates of infection. Literature cited CDC. 2002. Lyme Disease– United States 2000. MMWR 50:19-31. Adelson, M.E., Rao, R., Tilton, R. C., Cabets, K., Eskow, E., Fein, L., Occi, J., and Mordechai, E. 2004. Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis ticks collected in northern New Jersey. Journal of Clinical Microbiology 42(6): 2799- 2801. 546 bp A B CD http://cornellcollege.edu/biology/insects2005/images/ 4Ixodesscapularis.gif http://www.lyme.org/gallery/babe-miles1 http://en.wikipedia.org/wiki/Endocarditis http://www.marvistavet.com/html/body_ehrlich ia_infection_in_dogs.html Photo by Willy Burgdorfer E