Mass spectrometry-based proteomics of individual ticks demonstrated persistence of mammalian host blood components, including α-and β-globin chains, histones, and mitochondrial enzymes, in Ixodes scapularis and Amblyomma americanum ticks for months after molting. Residual host proteins may identify sources of infection for ticks.W ithout transovarial or venereal transmission, a vector-borne pathogen's persistence in nature depends on successful passage between >1 species of vertebrate reservoirs. For Lyme borreliosis in eastern North America, black-legged tick (Ixodes scapularis) larvae acquire Borrelia burgdorferi from a reservoir host during their fi rst blood meal. Infection persists through subsequent molts, and when a tick feeds for the second time as a nymph it may transmit infection to another competent reservoir or to a human. Reservoir hosts for B. burgdorferi are commonly white-footed mice but also include chipmunks, voles, shrews, and ground-foraging birds.When a tick-borne agent has multiple reservoir hosts, assigning relative contributions of each species to maintenance of the pathogen in the environment may be diffi cult. One approach is to capture animals, sample blood or tissue for evidence of infection, and examine embedded ticks for the microorganism (1,2). However, this approach is laborand resource-intensive, and sample sizes are limited. Greater statistical power could be attained with fewer resources if questing ticks were examined not only for infection but also for the source of the last blood meal because the tick would likely have acquired the infection from that vertebrate. If the tick were engorged, this would be straightforward with the PCR, as demonstrated in mosquitoes (3,4). However, host-seeking nymphal hard ticks are fl at because their last blood meals were months earlier. Use of PCR to identify DNA of vertebrate mitochondria in ticks has been reported (5,6), but results lacked full sensitivity (7,8).An alternative approach is to detect residual proteins from the blood meal. Uptake and retention of host immunoglobulin into the hemolymph of different species of ticks have been documented (9), and Venneström and Jensen found vertebrate actin in I. ricinus nymphs weeks after the molt (10). Given these observations, we hypothesized that suffi cient host proteins remained in fl at ticks for identifi cation of blood meal by using proteins instead of DNA.
The StudyWe used mass spectrometry (MS)-based proteomics, as described by Breci et al. and Koller et al. (11,12). Individual ticks or pools were pulverized after freezing in liquid nitrogen. Total proteins were precipitated in 95% ethanol at -20°C and recovered by centrifugation. Proteins of individual ticks were reduced with 100 mmol/L dithiothreitol, alkylated with 50 mmol/L iodoacetamide, digested with trypsin at a fi nal concentration of 0.01 μg/μL, and fi ltered through a C18 cartridge before being subjected to liquid chromatography (LC) with a 5%-50% acetonitrile gradient in 0.1% formic acid, followed by tandem MS (LC-MS/ MS; L...