Differential regulation of p53 function by the N-terminal ΔNp53 and Δ113p53 isoforms in zebrafish embryos.

BACKGROUND: The p53 protein family coordinates stress responses of cells and organisms. Alternative promoter usage and/or splicing of p53 mRNA gives rise to at least nine mammalian p53 proteins with distinct N- and C-termini which are differentially expressed in normal and malignant cells. The human N-terminal p53 variants contain either the full-length (FL), or a truncated (ΔN/Δ40) or no transactivation domain (Δ133) altogether. The functional consequences of coexpression of the different p53 isoforms are poorly defined. Here we investigated functional aspects of the zebrafish ΔNp53 ortholog in the context of FLp53 and the zebrafish Δ133p53 ortholog (Δ113p53) coexpressed in the developing embryo. RESULTS: We cloned the zebrafish ΔNp53 isoform and determined that ionizing radiation increased expression of steady-state ΔNp53 and Δ113p53 mRNA levels in zebrafish embryos. Ectopic ΔNp53 expression by mRNA injection caused hypoplasia and malformation of the head, eyes and somites, yet partially counteracted lethal effects caused by concomitant expression of FLp53. FLp53 expression was required for developmental aberrations caused by ΔNp53 and for ΔNp53-dependent expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A, p21, Cip1, WAF1). Knockdown of p21 expression markedly reduced the severity of developmental malformations associated with ΔNp53 overexpression. By contrast, forced Δ113p53 expression had little effect on ΔNp53-dependent embryonal phenotypes. These functional attributes were shared between zebrafish and human ΔNp53 orthologs ectopically expressed in zebrafish embryos. All 3 zebrafish isoforms could be coimmunoprecipitated with each other after transfection into Saos2 cells. CONCLUSIONS: Both alternative N-terminal p53 isoforms were expressed in developing zebrafish in response to cell stress and antagonized lethal effects of FLp53 to different degrees. However, in contrast to Δ113p53, forced ΔNp53 expression itself led to developmental defects which depended, in part, on p21 transactivation. In contrast to FLp53, the developmental abnormalities caused by ΔNp53 were not counteracted by concomitant expression of Δ113p53.

Coordinate control of cell cycle regulatory genes in zebrafish development tested by cyclin D1 knockdown with morpholino phosphorodiamidates and hydroxyprolyl-phosphono peptide nucleic acids.

During early zebrafish (Danio rerio) development zygotic transcription does not begin until the mid-blastula transition (MBT) 3 h after fertilization. MBT demarcates transition from synchronous short cell cycles of S and M phases exclusively to full cycles encompassing G1 and G2 phases. Transcriptional profiling and RT-PCR analyses during these phases enabled us to determine that this shift corresponds to decreased transcript levels of S/M phase cell cycle control genes (e.g. ccna2, ccnb1, ccnb2 and ccne) and increased transcript levels of ccnd1, encoding cyclin D1, and orthologs of p21 (p21-like) and retinoblastoma (Rb-like 1). To investigate the regulation of this process further, the translation of ccnd1 mRNA, a G1/S checkpoint control element, was impaired by microinjection of ccnd1-specific morpholino phosphorodiamidate (MO) 20mer or hydroxyprolyl-phosphono peptide nucleic acid (HypNA-pPNA) 16mer antisense oligonucleotides. The resulting downregulation of cyclin D1 protein resulted in microophthalmia and microcephaly, but not lethality. The phenotypes were not seen with 3-mismatch MO 20mers or 1-mismatch HypNA-pPNA 16mers, and were rescued by an exogenous ccnd1 mRNA construct with five mismatches. Collectively, these results indicate that transcription of key molecular determinants of asynchronous cell cycle control in zebrafish embryos commences at MBT and that the reduction of cyclin D1 expression compromises zebrafish eye and head development.

A preliminary investigation of Ehrlichia species in ticks, humans, dogs, and capybaras from Brazil.

A molecular epidemiologic investigation in two Brazilian states (Rondônia and São Paulo) was undertaken to determine if Ehrlichia species responsible for human and animal ehrlichioses in North America could be found in Brazilian vectors, potential natural mammalian reservoirs and febrile human patients with a tick bite history. Samples, including 376 ticks comprising 9 Amblyomma species, 29 capybara (Hydrochaeris hydrochaeris) spleens, 5 canine blood, and 75 human blood samples from febrile patients with history of tick bites were tested by a real-time PCR assay targeting a fragment of the Ehrlichia dsb gene. Ehrlichia DNA was not detected in any tick, capybara or human samples. In contrast, 4 out of 5 dogs contained Ehrlichia canis DNA in their blood, which were sequenced, representing the first report of E. canis infecting dogs in the Amazon region of Brazil. Further studies are needed to evaluate the presence of other agents of human and animal ehrlichioses in Brazil.

Loss of heparan N-sulfotransferase in diabetic liver: role of angiotensin II.

The basis for accelerated atherosclerosis in diabetes is unclear. Diabetes is associated with loss of heparan sulfate (HS) from the liver, which may impede lipoprotein clearance and thereby worsen atherosclerosis. To study hepatic HS loss in diabetes, we examined regulation of HS N-deacetylase/N-sulfotransferase-1 (NDST), a key enzyme in hepatic HS biosynthesis. Hepatic NDST mRNA, protein, and enzymatic activity were suppressed by >50% 2 weeks after induction of type 1 diabetes in rats. Treatment of diabetic rats with enalapril, an ACE inhibitor, had no effect on hyperglycemia or hepatic NDST mRNA levels, yet increased hepatic NDST protein and enzymatic activity. Similar results were obtained in diabetic animals treated with losartan, which blocks the type 1 receptor for angiotensin II (AngII). Consistent with these findings, diabetic livers exhibited increased ACE expression, and addition of AngII to cultured hepatoma cells reduced NDST activity and protein. We conclude that diabetes substantially suppresses hepatic NDST mRNA, protein, and enzymatic activity. AngII contributes to suppression of NDST protein and enzymatic activity, whereas mRNA suppression occurs independently. Suppression of hepatic NDST may contribute to diabetic dyslipidemia, and stimulation of NDST activity by AngII inhibitors may provide cardiovascular protection.

Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos.

PURPOSE: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance the radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. METHODS AND MATERIALS: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. RESULTS: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the "curly up" phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. CONCLUSIONS: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

Co-infection of white-tailed deer with multiple strains of Ehrlichia chaffeensis.

We investigated the effect of exposing deer to multiple strains of Ehrlichia chaffeensis that differed in number of tandem repeats in either the variable-length PCR target (VLPT) gene or 120 kDa antigen gene. We hypothesized that infection with one strain would provide immunity to infection with other strains of E. chaffeensis. All deer initially exposed to strain A (604-2) became PCR and culture positive by 10 days post-infection (DPI). Three deer infected with strain A and subsequently inoculated with strain B (623-4) became infected with strain B. Two deer infected with strain A and subsequently inoculated with strain C (125B) became infected with strain C. Of three deer, each infected with strain B and subsequently inoculated with strain C, one was PCR positive for strain C. Of three deer previously inoculated with both strains A and B, and subsequently inoculated with strain C, one showed delayed evidence of strain C. Western blot analysis demonstrated that deer sera reacted differently to antigens from each exposed strain. A complementary in vitro study demonstrated that exposure to two strains differing in VLPT repeats may lead to co-infection of DH82 cells. These results complement a previous study and further show that deer can become sequentially infected with up to three strains of E. chaffeensis. This suggests that competitive exclusion, a phenomenon described in related organisms such as Anaplasma marginale whereby infection with one strain precludes subsequent infection by a second, distinct strain of the same species, may not occur with E. chaffeensis.

Evaluation of white-tailed deer (Odocoileus virginianus) as natural sentinels for Anaplasma phagocytophilum.

Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis, can infect white-tailed deer (WTD; Odocoileus virginianus), and this species is a crucial host for adult Ixodes scapularis, the primary vector of A. phagocytophilum. The goal of this study was to determine the geographic distribution of A. phagocytophilum among WTD across a 19 state region and to evaluate the utility of WTD as natural sentinels. Serologic testing using the indirect fluorescent antibody (IFA) assay was conducted on WTD serum samples and molecular and xenodiagnostic tests were performed to confirm serologic results. The surveillance system was assessed through examination of vital attributes including WTD age and gender associations with serologic status, sample size adequacy for accurate infection status classification, and presence of the vector, I. scapularis. Six hundred thirty-three of 2,666 (24%) WTD in 17 states tested positive for antibodies (>or=128) when tested by IFA assay. Testing for p44 and/or 16S rRNA gene targets identified 73 (16%) PCR positive WTD among 458 animals tested, all of which originated from seropositive populations. Attempts to culture A. phagocytophilum from WTD were unsuccessful; however, xenodiagnostic mice inoculated with blood from 3 WTD became infected. Seroprevalence did not differ by deer age or gender; however, WTD

Wild bird mortality and West Nile virus surveillance: biases associated with detection, reporting, and carcass persistence.

Surveillance targeting dead wild birds, in particular American crows (Corvus brachyrhynchos), plays a critical role in West Nile virus (WNV) surveillance in the United States. Using crow decoy surrogates, detection and reporting of crow carcasses within urban and rural environments of DeKalb County, Georgia were assessed for potential biases that might occur in the county's WNV surveillance program. In each of two replicated trials, during July and September 2003, 400 decoys were labeled with reporting instructions and distributed along randomly chosen routes throughout designated urban and rural areas within DeKalb County. Information-theoretic methods were used to compare alternative models incorporating the effects of area and trial on probabilities of detection and reporting. The model with the best empirical support included the effects of area on both detection and reporting of decoys. The proportion of decoys detected in the urban area (0.605, SE=0.024) was approximately twice that of the rural area (0.293, SE=0.023), and the proportion of decoys reported in the urban area (0.273, SE=0.023) was approximately three times that of the rural area (0.103, SE=0.028). These results suggest that human density and associated factors can substantially influence dead crow detection and reporting and, thus, the perceived distribution of WNV. In a second and separate study, the persistence and fate of American crow and house sparrow (Passer domesticus) carcasses were assessed in urban and rural environments in Athens-Clarke, Madison, and Oconee counties, Georgia. Two replicated trials using 96 carcasses of each species were conducted during July and September 2004. For a portion of the carcasses, motion sensitive cameras were used to monitor scavenging species visits. Most carcasses (82%) disappeared or were decayed by the end of the 6-day study. Carcass persistence averaged 1.6 days in rural areas and 2.1 days in urban areas. We analyzed carcass persistence rates using a known-fate model framework in program MARK. Model selection based on Akaike's Information Criteria (AIC) indicated that the best model explaining carcass persistence rates included species and number of days of exposure; however, the model including area and number of days of exposure received approximately equal support. Model-averaged carcass persistence rates were higher for urban areas and for crow carcasses. Six mammalian and one avian species were documented scavenging upon carcasses. Dead wild birds could represent potential sources of oral WNV exposure to these scavenging species. Species composition of the scavenger assemblage was similar in urban and rural areas but "scavenging pressure" was greater in rural areas.

Noninvasive Testing in Emergency Department Patients with Low-Risk Chest Pain: Does the Evidence Support Current Guidelines?

Patients who present to the emergency department with chest pain but no evidence of ischemia on the electrocardiogram and negative cardiac markers are at very low risk. The newest American Heart Association/American College of Cardiology guidelines give noninvasive cardiac testing a IIa recommendation in this patient population. Here, we will review the existing literature that was cited in the American Heart Association/American College of Cardiology document, as well as several large, contemporary, comparative observational studies which were not included to address the following question: Do the benefits of noninvasive cardiac testing in this patient population outweigh the risks?

Comparison of the Effectiveness of Stress Echocardiography Versus Myocardial Perfusion Imaging in Patients Presenting to the Emergency Department With Low-Risk Chest Pain.

The aim of this study was to compare clinically relevant cardiovascular outcomes and downstream resource utilization associated with stress echocardiography (SE) and myocardial perfusion imaging (MPI) in emergency department patients with low-risk chest pain. This was a retrospective analysis of health insurance claims data for a national sample of privately insured patients over the period January 1 to December 31, 2011. Subjects were selected who presented to the emergency department with a primary or secondary diagnosis of chest pain and underwent either SE or MPI. The primary end points were the percentage of patients in each group who underwent downstream cardiac catheterization, revascularization, repeat noninvasive testing, return emergency department visit with chest pain, and hospitalization for myocardial infarction. The mean length of follow-up was 190 days in both groups. Overall, 48,202 patients or 24,101 propensity-matched pairs were included in the final analysis. Compared with SE, MPI was associated with significantly higher odds of subsequent cardiac catheterization (adjusted odds ratio [AOR] 2.15; 95% confidence interval [CI] 1.99 to 2.33) and revascularization procedures (AOR 1.58; 95% CI 1.36 to 1.85) and repeat emergency department visits (AOR 1.14; 95% CI 1.11 to 1.19). The odds of repeat testing and myocardial infarction did not differ between groups. The average cost of downstream care was significantly higher in the MPI group ($2,193.80 vs $1,631.10, p <0.0001). According to the a priori rules specified for this comparative analysis, SE is more effective than MPI for privately insured patients who present to the emergency department with chest pain. In conclusion, these findings demonstrate the importance of assessing diagnostic tests based on how they affect hard end points because identification of disease, in and of itself, may not confer any clinical advantage.

Spatial analysis of the distribution of Ehrlichia chaffeensis, causative agent of human monocytotropic ehrlichiosis, across a multi-state region.

Ehrlichia chaffeensis, which causes human monocytotrophic ehrlichiosis (HME), is an important emerging tick-borne pathogen in the southeastern and southcentral United States. The endemnicity probability of E. chaffeensis and, by implication, locations with risk for HME, was predicted by using two modeling methods. This is first large-scale study to use geospatial analyses to estimate the distribution of E. chaffeensis, and it was conducted using data from a prototypic surveillance system that used white-tailed deer as natural sentinels. Analyses included the E. chaffeensis serostatus for 563 counties from 18 states. Both kriging and logistic regression models provided very reliable portrayals of E. chaffeensis occurrence and predicted that E. chaffeensis distribution had good concordance with human case data. The integration of a deer surveillance system with geospatial analyses was useful in developing HME risk maps that will be useful for identifying high-risk areas for public health interventions such as prevention and control efforts.

Development of a new animal model of chronic mitral regurgitation in rats under transesophageal echocardiographic guidance.

Large animal models (dog and sheep) are often used for the investigation of the pathophysiology of chronic mitral regurgitation (MR). A major limitation of large animal models is cost. The aim of this study was to develop a new animal model of chronic MR. Left thoracotomy was performed in 34 rats. Under the guidance of transesophageal echocardiography, a fine needle was inserted into the left ventricle (LV) to damage the mitral leaflets and produce MR. Serial transthoracic echocardiography was performed to assess LV remodeling and function. Left atrial and LV diameters were significantly larger, and LV fractional shortening was lower in the MR group than in the sham group. The 150-day survival was 59% in the MR group and 100% in the sham group (P < .01). This new animal model of chronic MR may be used in the study of the pathophysiology of chronic MR and pharmacologic therapies.

Evidence of tick-borne organisms in mule deer (Odocoileus hemionus) from the western United States.

Free-ranging mule deer (MD; Odocoileus hemionus) from Arizona and California were tested for evidence of infection with several tick-borne pathogens, including species of Ehrlichia, Anaplasma, Babesia, and Borrelia. Of 125 mule deer tested from Arizona, 29 (23%) and 11 (9%) had antibodies reactive to E. chaffeensis and A. phagocytophilum by indirect immunofluorescent antibody testing, respectively; none of the six MD tested from California were seropositive. Using a commercial competitive ELISA kit, antibodies reactive to Anaplasma spp. were detected in 19 (15%) MD from Arizona and four of six (67%) MD from California. Polymerase chain reaction (PCR) testing for tick-borne pathogens was conducted on blood samples from 29 MD from Arizona and 11 MD from California. Twenty-two of 29 (75.9%) MD from Arizona had PCR evidence of infection with at least one tick-borne pathogen. We detected an Anaplasma sp. in 19 of 29 (65.5%) MD and a Babesia sp. in 10 of 29 (34%) MD. Sequencing of these amplicons indicated that the Anaplasma sp. was the same that had previously been detected in MD from California and the Babesia sp. was similar to one previously detected in a reindeer (Rangifer tarandus tarandus) from California. All of the California MD had evidence of infection with a tick-borne pathogen. Two different species of Anaplasma spp. were detected in MD from California, eight of of 11 MD were infected with an Anaplasma sp., and three of 11 MD were infected with A. ovis. This is the first report of a mule deer naturally infected with A. ovis. Ten of 11 MD from California were infected with a Babesia-like organism previously associated with human disease, and a single MD was PCR positive for Borrelia coriaceae, which has been associated with epizootic bovine abortion. Together, these data suggest that MD in northern Arizona and eastern California are exposed to several pathogens of human and veterinary importance.

Primary and secondary infection with Ehrlichia chaffeensis in white-tailed deer (Odocoileus virginianus).

White-tailed deer (Odocoileus virginianus) are the principal reservoir host for Ehrlichia chaffeensis, causative agent of human monocytic ehrlichiosis (HME). Because white-tailed deer maintain a long-term infection with E. chaffeensis and because deer can be naturally exposed to multiple strains of E. chaffeensis, we evaluated the response to secondary infection of E. chaffeensis in deer. For primary infection, six white-tailed deer were injected with 5.4 x 10(6) DH82 cells infected with the Arkansas strain of E. chaffeensis (Ark) and two control deer were injected with noninfected DH82 cells. On post-infection day 54, three E. chaffeensis (Ark) infected deer and one naive deer were injected with 4.2 x 10(6) cells infected with strain WTD-6045B E. chaffeensis, which differs from the Arkansas strain by number of nucleotide repeats in the variable length PCR target (VLPT) gene; three other Arkansas strain infected deer were injected with noninfected DH82 cells. All animals were monitored for 31 additional days. All deer in the primary infection became positive by PCR amplification of the 16S rRNA or VLPT genes and/or cell culture by DPI-8. PCR amplification of the VLPT gene on whole blood, cell culture, and tissues detected primary and/or secondary strains in all deer exposed to both primary and secondary strains; in one deer, the primary strain was cultured from the lymph node. Our culture results demonstrated that both strains were present; however, PCR detection suggests that the secondary strain may have been circulating in blood at higher levels. In conclusion, this study provides evidence that primary infection of deer with E. chaffeensis does not protect against subsequent exposure and confirms that deer can be simultaneously coinfected with at least two different strains of E. chaffeensis.

Detection of Ehrlichia spp. in raccoons (Procyon lotor) from Georgia.

Raccoons (Procyonis lotor) and opossums (Didelphis virginianus) acquired from six contiguous counties in the Piedmont physiographic region of Georgia were investigated for their potential role in the epidemiology of ehrlichial and anaplasmal species. Serum was tested by indirect fluorescent antibody (IFA) assay for the presence of antibodies reactive to Ehrlichia chaffeensis, E. canis, and Anaplasma phagocytophilum (HGA agent). Nested polymerase chain reaction (PCR) assay was used to test whole blood or white blood cell preparations for the presence of Ehrlichia and Anaplasma spp. 16S rRNA (rDNA) gene fragments. In addition, ticks were collected from these animals and identified. Twenty-three of 60 raccoons (38.3%) had E. chaffeensis-reactive antibodies (>1:64), 13 of 60 raccoons (21.7%) had E. canis-reactive antibodies, and one of 60 raccoons (1.7%) had A. phagocytophilum- reactive antibodies. A sequence confirmed E. canis product was obtained from one of 60 raccoons and a novel Ehrlichia-like 16S rDNA sequence was detected in 32 of 60 raccoons. This novel sequence was most closely related to an Ehrlichia-like organism identified from Ixodes ticks and rodents in Asia and Europe. Raccoons were PCR negative for E. chaffeensis and E. ewingii DNA. Five tick species, including Dermacentor variabilis, Amblyomma americanum, Ixodes texanus, I. cookei, and I. scapularis, were identified from raccoons and represent potential vectors for the ehrlichiae detected. Opossums (n = 17) were free of ticks and negative on all IFA and PCR assays. This study suggests that raccoons are potentially involved in the epidemiology of multiple ehrlichial organisms with known or potential public health and veterinary implications.

Comparative effectiveness of diagnostic testing strategies in emergency department patients with chest pain: an analysis of downstream testing, interventions, and outcomes.

IMPORTANCE: Patients presenting to the emergency department (ED) with chest pain whose evaluation for ischemia demonstrates no abnormalities receive further functional or anatomical studies for coronary artery disease; however, comparative evidence for the various strategies is lacking and multiple testing options exist. OBJECTIVE: To compare chest pain evaluation pathways based on their association with downstream testing, interventions, and outcomes for patients in EDs. DESIGN, SETTING, AND PARTICIPANTS: Retrospective analysis of health insurance claims data for a national sample of privately insured patients from January 1 to December 31, 2011. Individuals with a primary or secondary diagnosis of chest pain in the ED were selected and classified into 1 of 5 testing strategies: no noninvasive testing, exercise electrocardiography, stress echocardiography, myocardial perfusion scintigraphy, or coronary computed tomography angiography. MAIN OUTCOMES AND MEASURES: The proportion of patients in each group who received a cardiac catheterization, coronary revascularization procedure, or future noninvasive test as well as those who were hospitalized for an acute myocardial infarction (MI) during 7 and 190 days of follow-up. RESULTS: In 2011, there were 693 212 ED visits with a primary or secondary diagnosis of chest pain, accounting for 9.2% of all ED encounters. After application of the inclusion and exclusion criteria, 421 774 patients were included in the final analysis; 293 788 individuals did not receive an initial noninvasive test and 127 986 did, representing 1.7% of all ED encounters. Overall, the percentage of patients hospitalized with an MI was very low during both 7 and 190 days of follow-up (0.11% and 0.33%, respectively). Patients who did not undergo initial noninvasive testing were no more likely to experience an MI than were those who did receive testing. Compared with no testing, exercise electrocardiography, myocardial perfusion scintigraphy, and coronary computed tomography angiography were associated with significantly higher odds of cardiac catheterization and revascularization procedures without a concomitant improvement in the odds of experiencing an MI. CONCLUSIONS AND RELEVANCE: Patients with chest pain evaluated in the ED who do not have an MI are at very low risk of experiencing an MI during short- and longer-term follow-up in a cohort of privately insured patients. This low risk does not appear to be affected by the initial testing strategy. Deferral of early noninvasive testing appears to be reasonable.

An unexpected surgical complication of ventricular assist device implantation identified by transesophageal echocardiography: a case report.

Ventricular assist devices are used for circulatory support for patients with cardiogenic shock and refractory heart failure. Although early and late complications of left ventricular assist devices have been reported, we report a rare surgical complication of a left ventricular assist device implantation. This unexpected surgical complication was accurately identified by transesophageal echocardiography.

Evaluation of a prototype Ehrlichia chaffeensis surveillance system using white-tailed deer (Odocoileus virginianus) as natural sentinels.

The natural history of Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, includes the lone star tick (LST, Amblyomma americanum) as a vector and white-tailed deer (WTD; Odocoileus virginianus) as both a natural reservoir of E. chaffeensis and a major host of LST. The goal of the current study was to implement and evaluate a prototype surveillance system to delineate the geographic distribution of E. chaffeensis using WTD as natural sentinels. To accomplish this goal, serologic testing using the indirect immunofluorescent antibody (IFA) test was performed on WTD serum samples, and to confirm serologic results, polymerase chain reaction (PCR) assays and culture isolation were conducted. Considerations relevant to the applicability of a surveillance system utilizing WTD were analyzed (e.g., age and gender relationships to serologic status, adequacy of sample sizes needed to distinguish between uninfected and infected populations, presence of LST, and ability to detect stability and spread of E. chaffeensis in WTD populations). Of 3275 WTD serologically tested, 549 (47%) from 17 of 18 states had antibodies reactive to E. chaffeensis (IFA titer > or = 1:128). No difference between age groups or gender was noted with serologic testing, thus these variables would not be a concern for a surveillance system using WTD. Significantly more deer in younger age groups (< or = 1.5 yr) were PCR and culture positive, and 46% of 122 seropositive WTD populations were confirmed positive by PCR or culture isolation. A significant association between LST infestation and E. chaffeensis seroreactivity was noted. Furthermore, the surveillance system was able to detect stability of E. chaffeensis within WTD populations and also spread to new populations, both of which were associated with LST status. These data clearly demonstrate that WTD are useful as natural sentinels for this emerging human pathogen, and establish a prototypical framework for a WTD surveillance system.