The Spectrum of Erythema Migrans in Early Lyme Disease: Can We Improve Its Recognition?

Background and objective Diagnosis of early Lyme disease (LD) often relies on clinical recognition of the skin lesion, erythema migrans (EM), a diagnostic sign of disease when laboratory testing is insensitive. Because EM can present in morphologically distinct forms, its recognition by clinicians can be challenging. This study aimed to characterize the clinical spectrum of lesions in patients presenting with suspected early LD in an ambulatory care setting to identify features that might help clinicians to be better prepared to recognize EM lesions. Methods Images of lesions from 69 participants suspected to have early LD were retrospectively evaluated by a dermatologist and a family practitioner with expertise in early LD. Reviewers made determinations on the diagnoses and morphological features of lesions. Agreement between reviewers and associations among lesion types and participant demographics, symptomology, and laboratory evidence of infection were examined using the kappa statistic and contingency tables, respectively. Results Challenges in diagnosing EM were evident in our study: initial concordance between reviewers was moderate [kappa statistic (95% CI): 0.45 (0.245 - 0.657)]. The final classification included 35 lesions (51%) that were agreed to be EM; 23 lesions (30%) were considered to be possible early EM or tick bite reactions, and 11 (16%) were thought not to be EM, but rather other diagnoses, including ringworm, allergic contact dermatitis, and mosquito bites. Only two lesions (6%) were observed with a classic bull's eye or ring-within-a-ring pattern. Most EM lesions were uniform (51%), pink (74%), oval lesions (63%), with well-demarcated borders (92%). Early EM or tick bite reactions were typically <5 cm in size (74%), red (52%), round lesions (61%), with a punctum present (100%). Lesions thought not to be EM also tended to be pink or red (64%), round (55%), or uniform (45%) lesions, but also had raised (25%) or irregular borders (33%), which were not commonly observed in the reviewer-classified EM or tick bite reaction lesions. Participants with lesions classified as EM reported that they had the lesions for more days (p = 0.043) and reported more symptoms (p = 0.017) than participants with other lesions. Only 14 (20%) participants overall had positive laboratory evidence for LD; these included 13 (37%) of the participants with EM-classified lesions. Conclusions EM commonly occurs in forms that are not the classic bull's eye. Patients often present with lesions that may represent the very early stage of EM or tick bite reactions, and most patients will test negative on currently available laboratory tests, challenging clinicians in making an LD diagnosis or treatment decisions. Additional studies to further characterize the morphological features of EM and how variation in skin lesions may be perceived among clinicians would be helpful for developing guidelines on improving clinician recognition of EM.

Landscape features predict the current and forecast the future geographic spread of Lyme disease.

Lyme disease, the most prevalent vector-borne disease in North America, is increasing in incidence and geographic distribution as the tick vector, Ixodes scapularis, spreads to new regions. We re-construct the spatial-temporal invasion of the tick and human disease in the Midwestern US, a major focus of Lyme disease transmission, from 1967 to 2018, to analyse the influence of spatial factors on the geographic spread. A regression model indicates that three spatial factors-proximity to a previously invaded county, forest cover and adjacency to a river-collectively predict tick occurrence. Validation of the predictive capability of this model correctly predicts counties invaded or uninvaded with 90.6% and 98.5% accuracy, respectively. Reported incidence increases in counties after the first report of the tick; based on this modelled relationship, we identify 31 counties where we suspect I. scapularis already occurs yet remains undetected. Finally, we apply the model to forecast tick establishment by 2021 and predict 42 additional counties where I. scapularis will probably be detected based upon historical drivers of geographic spread. Our findings leverage resources dedicated to tick and human disease reporting and provide the opportunity to take proactive steps (e.g. educational efforts) to prevent and limit transmission in areas of future geographic spread.

Tickborne disease awareness and protective practices among U.S. Forest Service employees from the upper Midwest, USA.

BACKGROUND: People with occupations that require them to spend time working outdoors in suitable tick habitats are predicted to be at an increased risk for tick-borne diseases (TBDs). However, few studies have assessed the risks of outdoor employees in the United States. METHODS: We conducted a cross-sectional survey to collect data on exposure to ticks and TBD infections among U.S. Forest Service employees in a high TBD incidence region of northern Wisconsin, and to examine employee knowledge, attitudes, and practices (KAPs) regarding TBDs to help guide future education and prevention programs. Chi-square contingency tables, calculations of odds ratios, and logistic regression models were used to identify associations among self-reported employee factors, the proportion of correctly answered knowledge questions, their ranked concern for TBDs, adherence to practicing preventive behaviors, and willingness to pay for protective measures. RESULTS: Ninety-five employees completed the survey. Nearly all respondents (97%) reported recent tick exposure, with 27% reporting encountering 10 or more ticks per week during peak tick season. Employee knowledge of TBD was high (median score: 80% correct). Fifty-nine percent of respondents had high concern for TBDs, and there was high adherence to conducting body checks for ticks (83% reported always doing them), but only moderate use of tick repellents (24% reported always and 60% reported occasionally using). High concern for TBD (adjusted odds ratio (aOR) = 6.32 [95% confidence intervals, 1.97-20.28]), a history of TBD diagnosis (aOR = 5.88 [1.41-24.55]), and older age (≥ 46 years) (aOR = 3.29 [1.00-10.84]) were positively associated with high practice adherence. Respondents suggested they would be willing to pay for personal protective methods and get a hypothetical vaccine for Lyme disease, but not community-wide efforts to control ticks. CONCLUSIONS: Our study provides evidence that U.S. Forest Service employees in Wisconsin represent a high risk group for TBD, and despite relatively high TBD knowledge and engagement in tick protection activities, efforts are needed to reduce their risks for tick bites. More generally, our findings suggest that studies to better understand the factors related to the adoption and effectiveness of public health interventions are needed.

Spatio-Temporal Dynamics of Tick-Borne Diseases in North-Central Wisconsin from 2000-2016.

Lyme disease is a well-recognized public health problem in the USA, however, other tick-borne diseases also have major public health impacts. Yet, limited research has evaluated changes in the spatial and temporal patterns of non-Lyme tick-borne diseases within endemic regions. Using laboratory data from a large healthcare system in north-central Wisconsin from 2000-2016, we applied a Kulldorf's scan statistic to analyze spatial, temporal and seasonal clusters of laboratory-positive cases of human granulocytic anaplasmosis (HGA), babesiosis, and ehrlichiosis at the county level. Older males were identified as the subpopulation at greatest risk for non-Lyme tick-borne diseases and we observed a statistically significant spatial and temporal clustering of cases (p < 0.05). HGA risk shifted from west to east over time (2000-2016) with a relative risk (RR) ranging from 3.30 to 11.85, whereas babesiosis risk shifted from south to north and west over time (2004-2016) with an RR ranging from 4.33 to 4.81. Our study highlights the occurrence of non-Lyme tick-borne diseases, and identifies at-risk subpopulations and shifting spatial and temporal heterogeneities in disease risk. Our findings can be used by healthcare providers and public health practitioners to increase public awareness and improve case detection.

The Lyme Disease Biobank: Characterization of 550 Patient and Control Samples from the East Coast and Upper Midwest of the United States.

Lyme disease (LD) is an increasing public health problem. Current laboratory testing is insensitive in early infection, the stage at which appropriate treatment is most effective in preventing disease sequelae. The Lyme Disease Biobank (LDB) collects samples from individuals with symptoms consistent with early LD presenting with or without erythema migrans (EM) or an annular, expanding skin lesion and uninfected individuals from areas of endemicity. Samples were collected from 550 participants (298 cases and 252 controls) according to institutional review board-approved protocols and shipped to a centralized biorepository. Testing was performed to confirm the presence of tick-borne pathogens by real-time PCR, and a subset of samples was tested for Borrelia burgdorferi by culture. Serology was performed on all samples using the CDC's standard two-tiered testing algorithm (STTTA) for LD. LD diagnosis was supported by laboratory testing in 82 cases, including positive results by use of the STTTA, PCR, or culture or positive results by two enzyme-linked immunosorbent assays for cases presenting with EM lesion sizes of >5 cm. The remaining 216 cases had negative laboratory testing results. For the controls, 43 were positive by at least one of the tiers and 6 were positive by use of the STTTA. The results obtained with this collection highlight and reinforce the known limitations of serologic testing in early LD, with only 29% of individuals presenting with EM lesion sizes of >5 cm yielding a positive result using the STTTA. Aliquots of whole blood, serum, and urine from clinically characterized patients with and without LD are available to investigators in academia and industry for evaluation or development of novel diagnostic assays for LD, to continue to improve upon currently available methods.

Development and Validation of a Serologic Test Panel for Detection of Powassan Virus Infection in U.S. Patients Residing in Regions Where Lyme Disease Is Endemic.

Powassan virus (POWV) is an emerging tick-borne arbovirus presenting a public health threat in North America. POWV lineage II, also known as deer tick virus, is the strain of the virus most frequently found in Ixodes scapularis ticks and is implicated in most cases of POWV encephalitis in the United States. Currently, no commercial tests are available to detect POWV exposure in tick-borne disease (TBD) patients. We describe here the development and analytical validation of a serologic test panel to detect POWV infections. The panel uses an indirect enzyme immunoassay (EIA) to screen. EIA-positive samples reflex to a laboratory-developed, POWV-specific immunofluorescence assay (IFA). The analytical sensitivity of the test panel was 89%, and the limit of detection was a plaque reduction neutralization test (PRNT) titer of 1:20. The analytical specificity was 100% for the IgM assay and 65% for the IgG assay when heterologous-flavivirus-positive samples were tested. On samples collected from regions where Lyme disease is endemic, seroprevalence for POWV in TBD samples was 9.4% (10 of 106) versus 2% when tested with non-TBD samples (2 of 100, P = 0.034). No evidence of POWV infection was seen in samples collected from a region where Lyme disease was not endemic (0 of 22). This test panel provides a sensitive and specific platform for detecting a serologic response to POWV early in the course of infection when neutralizing antibodies may not be detectable. Combined with clinical history, the panel is an effective tool for identifying acute POWV infection. IMPORTANCE Approximately 100 cases of POWV disease were reported in the United States over the past 10 years. Most cases have occurred in the Northeast (52) and Great Lakes (45) regions (https://www.cdc.gov/powassan/statistics.html). The prevalence of POWV in ticks and mammals is increasing, and POWV poses an increasing threat in a greater geographical range. In areas of the Northeast and Midwest where Lyme disease is endemic, POWV testing is recommended for patients with a recent tick bite, patients with Lyme disease who have been treated with antibiotics, or patients with a tick exposure who have tested negative for Lyme disease or other tick-borne illnesses and have persistent symptoms consistent with posttreatment Lyme disease. Testing could also benefit patients with tick exposure and unexplained neurologic symptoms and chronic fatigue syndrome (CFS) patients with known tick exposure. Until now, diagnostic testing for Powassan virus has not been commercially available and has been limited to patients presenting with severe, neurologic complications. The lack of routine testing for Powassan virus in patients with suspected tick-borne disease means that little information is available regarding the overall prevalence of the virus and the full spectrum of clinical symptoms associated with infection. As Ixodes scapularis is the tick vector for Powassan virus and multiple other tick-borne pathogens, including the Lyme disease bacterium, Borrelia burgdorferi, the clinical presentations and long-term outcomes of Powassan virus infection and concurrent infection with other tick-borne disease pathogens remain unknown.

Clinical Presentation and Outcomes of Children With Human Granulocytic Anaplasmosis.

BACKGROUND: Adults with the tick-borne disease human granulocytic anaplasmosis (HGA) have a spectrum of acute febrile illnesses that, if untreated, might be severe. Clinical presentation and outcomes of children with HGA have been poorly described. METHODS: A retrospective analysis was conducted to determine the frequency, presentation, and outcomes of pediatric patients with HGA between 1994 and 2015 in a region of Wisconsin in which HGA is highly endemic. Patients with related International Classification of Diseases Ninth and Tenth Revision (ICD-9 and ICD-10, respectively) codes or positive HGA laboratory test results were evaluated and classified as having had confirmed, probable, or suspected HGA on the basis of the Centers for Disease Control and Prevention (CDC) case definition. The Fisher's exact and Wilcoxon rank-sum tests were used in statistical comparisons. RESULTS: Of 187 children identified with possible HGA, 17 (9%) had confirmed, 75 (40%) had probable, and 91 (49%) had suspected infections. The number of cases rose sharply in 2010 and has remained between 16 and 36 cases per year since that time. A minority of children with confirmed or probable infections had elevated liver transaminase levels (33%), leukopenia (24%), thrombocytopenia (17%), or anemia (8%); 6 (7%) of these children required hospitalization. Children with evidence of concurrent HGA and Lyme disease (27% of confirmed or probable cases) had a higher risk of hospitalization (odds ratio, 6.55 [95% confidence interval, 1.11-38.78]). None of these children had life-threatening disease or died. CONCLUSIONS: Evidence suggests that the frequency of HGA in children is increasing. Although most children had mild disease, doxycycline remains the treatment of choice, because outcome data for children without treatment remains limited.

Risk Factors for Severe Infection, Hospitalization, and Prolonged Antimicrobial Therapy in Patients with Babesiosis.

Babesiosis is an emerging tick-borne disease transmitted by the hard tick Ixodes scapularis, which also transmits Lyme disease. Better gradation of prognostic indicators are needed to determine which patients may develop serious complications requiring hospitalization, and to provide early guidance on appropriate therapy. In this study, we evaluated 128 patients with smear or real time polymerase chain reaction-confirmed Babesia microti infections over a period of 16 years. Patients with asplenia or immunocompromising conditions were more likely to have severe infection (P < 0.01), require hospitalization (P < 0.01), or receive prolonged courses of antimicrobials (P < 0.01). Nausea or vomiting (P < 0.01) and diarrhea (P < 0.01) along with hyperbilirubinemia (P < 0.01) were predictive of severe infection, hospitalization, and prolonged antimicrobial therapy. Patients with concurrent Lyme disease were less likely to require hospitalization and had similar severity of disease and length of antibiotic treatment compared with those without Lyme disease.

Serologic Evidence of Powassan Virus Infection in Patients with Suspected Lyme Disease1.

Powassan virus (POWV) lineage II is an emerging tickborne flavivirus with an unknown seroprevalence in humans. In a Lyme disease-endemic area, we examined the seroreactivity to POWV in 2 patient cohorts and described the clinical features of the POWV-seroreactive patients. POWV disease might be less neuroinvasive than previously thought.

T2 Magnetic Resonance Assay-Based Direct Detection of Three Lyme Disease-Related Borrelia Species in Whole-Blood Samples.

In early Lyme disease (LD), serologic testing is insensitive and seroreactivity may reflect active or past infection. In this study, we evaluated a novel assay for the direct detection of three species of Borrelia spirochetes in whole blood. The T2 magnetic resonance (T2MR) assay platform was used to amplify Borrelia DNA released from intact spirochetes and to detect amplicon. Analytical sensitivity was determined from blood spiked with known concentrations of spirochetes, and the assay's limit of detection was found to be in the single-cell-per-milliliter range: 5 cells/ml for B. afzelii and 8 cells/ml for Borrelia burgdorferi and Borrelia garinii Clinical samples (n = 66) from confirmed or suspected early LD patients were also analyzed. B. burgdorferi was detected using T2MR in 2/2 (100%) of blood samples from patients with confirmed early LD, based on the presence of erythema migrans and documentation of seroconversion or a positive real-time blood PCR. T2MR detected B. burgdorferi in blood samples from 17/54 (31%) of patients with probable LD, based on the presence of erythema migrans without documented seroconversion or of documented seroconversion in patients with a compatible clinical syndrome but without erythema migrans. Out of 21 clinical samples tested by real-time PCR, only 1 was positive and 13 were negative with agreement with T2MR. An additional 7 samples that were negative by real-time PCR were positive with T2MR. Therefore, T2MR enables a low limit of detection (LoD) for Borrelia spp. in whole blood samples and is able to detect B. burgdorferi in clinical samples.

Pro-inflammatory immune responses are associated with clinical signs and symptoms of human anaplasmosis.

Human anaplasmosis (HA) is an emerging tick-borne disease that may present as a mild flu-like illness or a life threatening, sepsis-like condition. Although disease severity is hypothesized to relate to immunopathology and immune dysfunction in humans, studies to directly measure immune responses in infected humans have been very limited. We quantified cytokines in 80 confirmed HA patients using a multiplex chemiluminescence immunoassay system and compared similarly measured responses in 1000 control subjects. Pro-inflammatory cytokines were significantly elevated in HA patients (all seven p<0.0001). Interferon gamma (IFN-γ) concentrations were particularly high, with average concentrations 7.8 times higher in the HA patients than the controls. A subset of cytokines consisting of IL-1ß, IL-8, IL-6, TNF-α, and IL-10 was also coordinately high and significantly associated with severity of thrombocytopenia in HA patients. Patients with infections in the very acute stage (≤ 4 days ill) tended to have the highest IFN-γ, IL-12p70, and IL-2 levels. Higher concentrations of IL-13 and IL-5 were associated with diarrhea and vomiting. Our findings support a pathophysiological role for a pro-inflammatory response in HA, especially with regard to the modulation of hematopoiesis and subsequent hematopoietic complications.

Serologic screening of United States blood donors for Babesia microti using an investigational enzyme immunoassay.

BACKGROUND: The tick-borne pathogen Babesia microti has become recognized as the leading infectious risk associated with blood transfusion in the United States, yet no Food and Drug Administration-licensed screening tests are currently available to mitigate this risk. The aim of this study was to evaluate the performance of an investigational enzyme immunoassay (EIA) for B. microti as a screening test applied to endemic and nonendemic blood donor populations. STUDY DESIGN AND METHODS: The study aimed to test 20,000 blood donors from areas of the United States considered endemic for B. microti and 10,000 donors from a nonendemic area with the investigational B. microti EIA. Repeat-reactive samples were retested by polymerase chain reaction (PCR), blood smear, immunofluorescent assay (IFA), and immunoblot assay. In parallel, serum samples from symptomatic patients with confirmed babesiosis were tested by EIA, IFA, and immunoblot assays. RESULTS: A total of 38 of 13,757 (0.28%) of the donors from New York, 7 of 4583 (0.15%) from Minnesota, and 11 of 8363 (0.13%) from New Mexico were found repeat reactive by EIA. Nine of the 56 EIA repeat-reactive donors (eight from New York and one from Minnesota) were positive by PCR. The specificity of the assay in a nonendemic population was 99.93%. Among IFA-positive clinical babesiosis patients, the sensitivity of the assay was 91.1%. CONCLUSION: The B. microti EIA detected PCR-positive, potentially infectious blood donors in an endemic population and exhibited high specificity among uninfected and unexposed individuals. The EIA promises to provide an effective tool for blood donor screening for B. microti in a format amenable to high-throughput and cost-effective screening.

Ecology and Epidemiology of Lyme Borreliosis.

Lyme borreliosis is a zoonotic, tick-borne disease that infects humans worldwide. The disease is currently recognized as the most common vector-borne disease in Europe and North America. Disease is caused by several genospecies of the Borrelia burgdorferi sensu lato complex. Humans are at high risk of infection in regions where highly competent reservoirs are the primary hosts for the subadult stages of the tick, in contrast to regions where less competent or refractory animals feed ticks. Human infections are also most frequently associated with spring and summer months when the nymph stage of the tick is active.

Predator diversity, intraguild predation, and indirect effects drive parasite transmission.

Humans are altering biodiversity globally and infectious diseases are on the rise; thus, there is interest in understanding how changes to biodiversity affect disease. Here, we explore how predator diversity shapes parasite transmission. In a mesocosm experiment that manipulated predator (larval dragonflies and damselflies) density and diversity, non-intraguild (non-IG) predators that only consume free-living cercariae (parasitic trematodes) reduced metacercarial infections in tadpoles, whereas intraguild (IG) predators that consume both parasites and tadpole hosts did not. This likely occurred because IG predators reduced tadpole densities and anticercarial behaviors, increasing per capita exposure rates of the surviving tadpoles (i.e., via density- and trait-mediated effects) despite the consumption of parasites. A mathematical model demonstrated that non-IG predators reduce macroparasite infections, but IG predation weakens this "dilution effect" and can even amplify parasite burdens. Consistent with the experiment and model, a wetland survey revealed that the diversity of IG predators was unrelated to metacercarial burdens in amphibians, but the diversity of non-IG predators was negatively correlated with infections. These results are strikingly similar to generalities that have emerged from the predator diversity-pest biocontrol literature, suggesting that there may be general mechanisms for pest control and that biocontrol research might inform disease management and vice versa. In summary, we identified a general trait of predators--where they fall on an IG predation continuum--that predicts their ability to reduce infections and possibly pests in general. Consequently, managing assemblages of predators represents an underused tool for the management of human and wildlife diseases and pest populations.

A clinical, diagnostic, and ecologic perspective on human anaplasmosis in the Upper Midwest.

INTRODUCTION: Human anaplasmosis caused by the bacterial pathogen Anaplasma phagocytophilum was first discovered in the Upper Midwest in 1990. Since that time the number of cases in the region has steadily increased, such that today, the pathogen rivals that of Lyme disease in causing human tick-borne-related illness. OBJECTIVE: We provide an overview of the biology, clinical characteristics, and epidemiology of the disease in the Upper Midwest and discuss currently available diagnostic methods. FINDINGS: Rapid differentiation of anaplasmosis from other acute febrile illnesses and targeted treatment are important for preventing severe disease and potentially fatal outcomes in infected individuals. Beyond blood smear analysis and serology, the development of real-time polymerase chain reaction (PCR) assays for clinical use holds promise in improving our ability to make rapid diagnoses and to differentiate A phagocytophilum infections from those produced by closely related Ehrlichia pathogens, which are also present in the region. CONCLUSION: Continuing expansion of the range of the black-legged tick (Ixodes scapuloris), the principal vector of the disease, into areas heavily populated or visited by humans in the region likely will result in this pathogen becoming an even greater burden on human health. Efforts are needed to better characterize the current geographic distribution of human Anaplasma and Ehrlichia cases to identify emerging foci and to better understand the enzootic cycles that maintain the pathogens in the region. Improved diagnostics may assist with such efforts.

Effects of low-temperature flea maintenance on the transmission of Yersinia pestis by Oropsylla montana.

Yersinia pestis, the causative agent of plague, is primarily a rodent-associated, flea-borne zoonosis maintained in sylvatic foci throughout western North America. Transmission to humans is mediated most commonly by the flea vector Oropsylla montana and occurs predominantly in the southwestern United States. With few exceptions, previous studies showed O. montana to be an inefficient vector at transmitting Y. pestis at ambient temperatures, particularly when such fleas were fed on susceptible hosts more than a few days after ingesting an infectious blood meal. We examined whether holding fleas at subambient temperatures affected the transmissibility of Y. pestis by this vector. An infectious blood meal containing a virulent Y. pestis strain (CO96-3188) was given to colony-reared O. montana fleas. Potentially infected fleas were maintained at different temperatures (6°C, 10°C, 15°C, or 23°C). Transmission efficiencies were tested by allowing up to 15 infectious fleas to feed on each of 7 naïve CD-1 mice on days 1-4, 7, 10, 14, 17, and 21 postinfection (p.i.). Mice were monitored for signs of infection for 21 days after exposure to infectious fleas. Fleas held at 6°C, 10°C, and 15°C were able to effectively transmit at every time point p.i. The percentage of transmission to naïve mice by fleas maintained at low temperatures (46.0% at 6°C, 71.4% at 10°C, 66.7% at 15°C) was higher than for fleas maintained at 23°C (25.4%) and indicates that O. montana fleas efficiently transmit Y. pestis at low temperatures. Moreover, pooled percent per flea transmission efficiencies for flea cohorts maintained at temperatures of 10°C and 15°C (8.67% and 7.87%, respectively) showed a statistically significant difference in the pooled percent per flea transmission efficiency from fleas maintained at 23°C (1.94%). This is the first comprehensive study to demonstrate efficient transmission of Y. pestis by O. montana fleas maintained at temperatures as low as 6°C. Our findings further contribute to the understanding of plague ecology in temperate climates by providing support for the hypothesis that Y. pestis is able to overwinter within the flea gut and potentially cause infection during the following transmission season. The findings also might hold implications for explaining the focality of plague in tropical regions.

Changing socioeconomic indicators of human plague, New Mexico, USA.

Socioeconomic indicators associated with temporal changes in the distribution of human plague cases in New Mexico were investigated for 1976-2007. In the 1980s, cases were more likely in census block groups with poor housing conditions, but by the 2000s, cases were associated with affluent areas concentrated in the Santa Fe-Albuquerque region.

Effects of temperature on the transmission of Yersinia Pestis by the flea, Xenopsylla Cheopis, in the late phase period.

BACKGROUND: Traditionally, efficient flea-borne transmission of Yersinia pestis, the causative agent of plague, was thought to be dependent on a process referred to as blockage in which biofilm-mediated growth of the bacteria physically blocks the flea gut, leading to the regurgitation of contaminated blood into the host. This process was previously shown to be temperature-regulated, with blockage failing at temperatures approaching 30°C; however, the abilities of fleas to transmit infections at different temperatures had not been adequately assessed. We infected colony-reared fleas of Xenopsylla cheopis with a wild type strain of Y. pestis and maintained them at 10, 23, 27, or 30°C. Naïve mice were exposed to groups of infected fleas beginning on day 7 post-infection (p.i.), and every 3-4 days thereafter until day 14 p.i. for fleas held at 10°C, or 28 days p.i. for fleas held at 23-30°C. Transmission was confirmed using Y. pestis-specific antigen or antibody detection assays on mouse tissues. RESULTS: Although no statistically significant differences in per flea transmission efficiencies were detected between 23 and 30°C, efficiencies were highest for fleas maintained at 23°C and they began to decline at 27 and 30°C by day 21 p.i. These declines coincided with declining median bacterial loads in fleas at 27 and 30°C. Survival and feeding rates of fleas also varied by temperature to suggest fleas at 27 and 30°C would be less likely to sustain transmission than fleas maintained at 23°C. Fleas held at 10°C transmitted Y. pestis infections, although flea survival was significantly reduced compared to that of uninfected fleas at this temperature. Median bacterial loads were significantly higher at 10°C than at the other temperatures. CONCLUSIONS: Our results suggest that temperature does not significantly effect the per flea efficiency of Y. pestis transmission by X. cheopis, but that temperature is likely to influence the dynamics of Y. pestis flea-borne transmission, perhaps by affecting persistence of the bacteria in the flea gut or by influencing flea survival. Whether Y. pestis biofilm production is important for transmission at different temperatures remains unresolved, although our results support the hypothesis that blockage is not necessary for efficient transmission.

Effects of wetland vs. landscape variables on parasite communities of Rana pipiens: links to anthropogenic factors.

The emergence of several diseases affecting amphibian populations worldwide has prompted investigations into determinants of the occurrence and abundance of parasites in frogs. To understand the spatial scales and identify specific environmental factors that determine risks of parasitism in frogs, helminth communities in metamorphic frogs of the northern leopard frog (Rana pipiens) were examined in relation to wetland and landscape factors at local (1 km) and regional (10 km) spatial extents in an agricultural region of Minnesota (USA) using regression analyses, ordination, and variance partitioning techniques. Greater amounts of forested and woody wetland habitats, shorter distances between woody wetlands, and smaller-sized open water patches in surrounding landscapes were the most consistently positive correlates with the abundances, richness, and diversity of helminths found in the frogs. Wetland and local landscape variables were suggested as most important for larval trematode abundances, whereas local and regional landscape variables appeared most important for adult helminths. As previously reported, the sum concentration of atrazine and its metabolite desethylatrazine, was the strongest predictor of larval trematode communities. In this report, we highlight the additional influences of landscape factors. In particular, our data suggest that anthropogenic activities that have resulted in the loss of the availability and connectivity of suitable habitats in the surrounding landscapes of wetlands are associated with declines in helminth richness and abundance, but that alteration of wetland water quality through eutrophication or pesticide contamination may facilitate the transmission of certain parasite taxa when they are present at wetlands. Although additional research is needed to quantify the negative effects of parasitism on frog populations, efforts to reduce inputs of agrochemicals into wetlands to limit larval trematode infections may be warranted, given the current high rates of amphibian declines and extinction events.

Effects of temperature on early-phase transmission of Yersina pestis by the flea, Xenopsylla cheopis.

Sharp declines in human and animal cases of plague, caused by the bacterium Yersinia pestis (Yersin), have been observed when outbreaks coincide with hot weather. Failure of biofilm production, or blockage, to occur in the flea, as temperatures reach 30 degrees C has been suggested as an explanation for these declines. Recent work demonstrating efficient flea transmission during the first few days after fleas have taken an infectious blood meal, in the absence of blockage (e.g., early-phase transmission), however, has called this hypothesis into question. To explore the potential effects of temperature on early-phase transmission, we infected colony-reared Xenopsylla cheopis (Rothchild) fleas with a wild-type strain of plague bacteria using an artificial feeding system, and held groups of fleas at 10, 23, 27, and 30 degrees C. Naive Swiss Webster mice were exposed to fleas from each of these temperatures on days 1-4 postinfection, and monitored for signs of infection for 21 d. Temperature did not significantly influence the rates of transmission observed for fleas held at 23, 27, and 30 degrees C. Estimated per flea transmission efficiencies for these higher temperatures ranged from 2.32 to 4.96% (95% confidence interval [CI]: 0.96-8.74). In contrast, no transmission was observed in mice challenged by fleas held at 10 degrees C (per flea transmission efficiency estimates, 0-1.68%). These results suggest that declines in human and animal cases during hot weather are not related to changes in the abilities of X. cheopis fleas to transmit Y. pestis infections during the early-phase period. By contrast, transmission may be delayed or inhibited at low temperatures, indicating that epizootic spread of Y. pestis by X. cheopis via early-phase transmission is unlikely during colder periods of the year.