Developing Multiple Lines of Evidence to Decrease Drainage-to-Surface Area Ratio for Effective Stormwater Control Sizing Using Bioretention.

Bioretention units were constructed at the US Environmental Protection Agency's Edison Environmental Center to evaluate drainage-to-surface runoff ratio for sizing of bioretention stormwater controls. Three sizes of hydraulically isolated bioretention units were tested in duplicate with changes in aspect ratio of length from inlet wall by doubling successive length from smallest (3.7 m) to largest (14.9 m) while width remained the same (7.1 m). The watershed areas were nominally the same, resulting in watershed-to-surface area ratios of 5.5:1 for largest duplicate units, 11:1 for the middle units, and 22:1 for the smallest. Each unit was instrumented for continuous monitoring with water content reflectometers (WCRs) and thermistors with data collected since November 2009. The bioretention units were filled with planting media initially comprising 90% sand and 10% sphagnum peat moss by volume and approximately 99% and 1%, respectively, by weight. These units were then planted between May and November of 2010 with a variety of native grasses, perennials, shrubs, and trees that were tolerant to inundation, drought and salt. In late 2012, a survey of the shrubs planted in these bioretention units was performed. The published results of the combined analyses of moisture content, rainfall, and size of shrubs indicated that the smaller units had superior shrub growth due to the more frequent saturation of the root zone as measured by WCR, while the plants in the largest units, particularly away from front wall where runoff entered, potentially relied on direct rainfall only. Starting in 2017, additional monitoring was performed in these units, including chemistry analysis by loss on ignition and total phosphorus of the engineered planting media and an additional survey of the plants. As in the previous study, plants did better in the medium (11:1) and small (22:1) bioretention units than in the largest units (5.5:1), and there was greater buildup of organic matter and phosphorus in the smaller units. One species of grass that dominated the two largest bioretention units away from the inlet was drought tolerant, which indicated that plants in these units relied on rainfall rather than stormwater runoff. Oversized units did not completely use the stromwater control volume, and many of the other original plantings grew slower or were less widespread in comparison to plantings in that smaller units that flooded more frequently and achieved greater growth. Practical Applications: Defining the size of stormwater controls can be difficult because there are often multiple objectives imposed on the final design of these structures, including safety and flooding. Results presented here would indicate that if the objective is to create a bioretention area with healthy vegetation, undersized controls may be acceptable because undersized infiltrating controls will have healthier plantings and infiltrate throughout the storm. For municipalities, this means that rights of way previously thought to be too small to use for infiltrative stormwater controls may be converted to such a purpose. This does not free municipalities from stormwater systems that address flooding and safety design objectives, but demonstrates that increasing plantings in the municipal right of way could help to address stormwater as well as other objectives, like greenhouse gas emissions, urban heat island reduction, and clean air. Distributed bioretention controls that capture part or all the runoff of the smaller, most frequent rainfall events should be incorporated throughout municipalities and into their overall stormwater control systems. If clogging by runoff is a concern, roof runoff may be more appropriate for bioretention, or other measures such as sediment capture or increased maintenance may need to be performed.

Seasonal variation in indicator organisms infiltrating from permeable pavement parking lots at the Edison Environmental Center, New Jersey.

Four types of permeable pavements were monitored at the Edison Environmental Center in Edison, New Jersey, for three water quality indicator organisms consisting of fecal coliform, enterococci, and Escherichia coli. This study expands a previously published result based on less than a year of available data. The current study reflects nearly 5 years of data collection with efforts focusing on collection of data in all four seasons to analyze seasonal effects and to understand the effects of pH on infiltrate concentrations. All three indicators were detected in infiltrates from all four permeable surfaces and as well as asphalt and roof runoff. Seasonally, the infiltrate during winter had fewer detections and lower enumerations and was most often significantly different than surface infiltrate and runoff for the other seasons. More significant concentration reductions were observed in summer and fall, and the lowest reduction was observed in winter. Pervious Asphalt treatment removed the most microorganisms for all three indicator organisms. A permeable interlocking concrete pavement (PICP) that was a replacement for pervious concrete during the study performed better than the original PICP most likely due to smaller gap spacing (8 mm compared to 12.7 mm) and correspondingly smaller specified surface aggregate compared to the original PICP. Percent concentration removal reductions based on geometric means were 89% or greater for PC, PA, and PICP for fecal coliform; 75% or better for PC, PA and PICP for E. coli; and 95% or greater for PC and PA for enterococci, while there were no annual removals for enterococci for original or new PICP nor removals for E. coli for original PICP and minimal removal for fecal coliform for original PICP. The major sources of fecal indicators in the stormwater runoff were most likely from the feces of deer, geese, and other wild animals. PRACTITIONER POINTS: The infiltrate during winter had fewer detections and lower enumerations and was most often significantly different than surface infiltrate and runoff for the other seasons. More significant concentration reductions were observed in summer and fall, and the lowest reduction was observed in winter. Pervious Asphalt treatment removed the most microorganisms for all three indicator organisms.

Predicting location and evaluating progression of clogging in a permeable pavement parking lot.

In 2009, a permeable pavement research and demonstration site was constructed at the Edison Environmental Center, Edison, NJ. Infiltration testing of three original permeable parking rows through August 2012 indicated that clogging occurred along the upgradient edge of these pavements from runoff that drained from adjacent impermeable driving lanes. A subsequent infiltration testing data collection effort from April 2017 through March 2020 focused on permeable interlocking concrete pavers (PICP) that replaces one of the original permeable surfaces. While the original infiltration study through 2012 used random locations throughout the permeable parking rows, the newer study targeted upgradient edge to identify where clogging would occur. Testing locations along the upgradient edge were selected based on a high-resolution survey (HRS) of the parking lot performed in December 2014. The HRS identified three low spots along the upgradient edge that eventually clogged in the new PICP infiltration study. The HRS may assist with maintenance routines. The newer study also supports the conclusion of the earlier study with regards to truncating the infiltration testing method, particularly for maintenance assessments.

Borrelia burgdorferi Antibody Test Results in Dogs Administered 4 Different Vaccines.

Vaccines against Borrelia burgdorferi are administered frequently to dogs in areas endemic for the infection. These vaccines produce an antibody response to spirochetal proteins that cross-react in many antibody tests, including immunofluorescence assay, Western blot, and whole cell ELISA used to document exposure to B. burgdorferi. The purpose of this study was to determine whether the in-clinic C6 ELISA assay (SNAP® 4Dx® Plus; IDEXX Laboratories, Inc., Westbrook, Maine) and the quantitative format C6 ELISA (Lyme Quant C6® Antibody Test, IDEXX Laboratories, Inc., Westbrook, ME) react to sera from dogs that have been vaccinated with 1 of 4 different commercially available B. burgdorferi vaccines. Four groups of 3 dogs each were each administered one of the 4 vaccines and sera evaluated over time by indirect fluorescent antibody assay, western blot immunoassay, the in-clinic C6 ELISA assay, and the quantitative format C6 ELISA. While all dogs developed B. burgdorferi antibodies detectable by indirect fluorescent antibody assay and western blot immunoassay after vaccination, none of the samples were positive in either of the C6 peptide-based assays. Based on these results, positive anti-C6 antibody results in client-owned dogs are likely to reflect exposure to B. burgdorferi rather than vaccination.

Development of a dehydrated fortified food base from fermented milk and parboiled wheat, and comparison of its composition and reconstitution behavior with those of commercial dried dairy-cereal blends.

Dehydrated blends of milk and cereal are reconstituted and consumed as a nutritious soup or porridge in many regions; the composition and reconstitution behavior of the blends are likely to impact on nutritional quality and consumer acceptability of the soup/porridge. Experimental samples of dried fermented milk-bulgur wheat blend (FMBW) and commercial samples of dried dairy-cereal blends, namely kishk, tarhana, and super cereal plus corn-soy blend (SCpCSB) were compared for composition, color, water sorption, and reconstitution characteristics. FMBW blends had higher contents of protein, Ca, lactose and lactic acid, lower levels of salt (NaCl) and Fe, and a lighter, more-yellow color (higher L* and b*-color co-ordinates) than tarhana or kishk. Compared with SCpCSB, FMBW had numerically higher levels of protein, lactose, and lactic acid, lower levels of Ca, Fe, Zn, and Mg, and lower pH. Tarhana had highest mean levels of starch, and on reconstitution (133 g/kg) had highest water holding capacity, viscosity during pasting and cooling, yield stress (σ 0), consistency coefficient (K), and viscosity on shearing from 20 to 120 s-1 at 60°C. Reconstituted FMBW, kishk, and SCpCSB had similar pasting and flow behavior properties. Overall, the composition (starch, protein, Ca, Mg), pasting and flow behavior characteristics of FMBW were closer to those SCpCSB and kishk than to tarhana. The results suggest that the FMBW powder, on appropriate supplementation with Ca, Fe, Zn and Mg, could be used for the development of customized fortified blended foods for specific groups.

Organism Detection in Permeable Pavement Parking Lot Infiltrates at the Edison Environmental Center, New Jersey.

Three types of permeable pavements were monitored at the Edison Environmental Center in Edison, New Jersey, for indicator organisms such as fecal coliform, enterococci, and Escherichia coli. Results showed that porous asphalt had a much lower concentration in monitored infiltrate compared to pervious concrete and permeable interlocking concrete pavers; concentrations of monitored organisms in infiltrate from porous asphalt were consistently below the bathing water quality standard and actually had limited detection. Fecal coliform and enterococci exceeded bathing water quality standards more than 72 and 34% of the time for permeable interlocking concrete pavers and pervious concrete, respectively. Concentration reductions greater than 90% were observed for all three indicator organisms for porous asphalt and fecal coliform and E. coli for pervious concrete when compared to runoff values, while permeable interlocking concrete pavers only had a modest (39%) observable reduction for E. coli only. The near absence of indicator organisms observed in the porous asphalt infiltrate may be due to the high pH potentially due to asphalt processing. Neither rain intensity nor temperature was demonstrated to have an observable effect in both concentrations of organisms and performance of permeable pavement; but this may due to the limitations of the dataset consisting of 16 events over an 8-month period.

Concurrent iron and zinc deficiencies in lactating mothers and their children 6-23 months of age in two agro-ecological zones of rural Ethiopia.

PURPOSE: The aim of this study is to examine the co-occurrences of low serum ferritin and zinc and anaemia among mothers and their children in two agro-ecological zones of rural Ethiopia. METHODS: Data were collected from 162 lactating mothers and their breast fed children aged 6-23 months. The data were collected via a structured interview, anthropometric measurements, and blood tests for zinc, ferritin and anaemia. Correlation, Chi-square and multivariable analysis were used to determine the association between nutritional status of mothers and children, and agro-ecological zones. RESULTS: Low serum levels of iron and zinc, anaemia and iron deficiency anaemia were found in 44.4, 72.2, 52.5 and 29.6% of children and 19.8, 67.3, 21.8, 10.5% of mothers, respectively. There was a strong correlation between the micronutrient status of the mothers and the children for ferritin, zinc and anaemia (p < 0.005). Deficiency in both zinc and ferritin and one of the two was observed in 19.1, and 53.7% of the mothers and 32.7 and 46.3%, of their children, respectively. In the 24 h before the survey, 82.1% of mothers and 91.9% of their infants consumed foods that can decrease zinc bioavailability while only 2.5% of mothers and 3.7% of their infants consumed flesh foods. CONCLUSION: This study shows that micronutrient deficiencies were prevalent among lactating mothers and their children, with variation in prevalence across the agro-ecological zones. This finding calls for a need to design effective preventive public health nutrition programs to address both the mothers' and their children's needs.

Sensitivity and specificity levels of two rapid assays for antibodies to Anaplasma spp. in dogs.

Canine anaplasmosis is a tick-borne disease of dogs that results following infection with Anaplasma phagocytophilum or Anaplasma platys. The SNAP 4Dx Plus test (IDEXX Laboratories) and the VetScan Canine Anaplasma Rapid test (Abaxis) are commercial in-house rapid tests for the detection of antibody to these 2 antigenically related Anaplasma species. We evaluated 2 tests using serum and whole blood samples obtained from reference laboratories and veterinary hospitals. Samples were obtained from regions of the country known to be habitats of the primary tick vectors. The A. phagocytophilum sample set comprised 236 dog sera from the northeastern and midwestern United States; the A. platys sample set comprised 179 sera from dogs living in the southwestern United States. An indirect immunofluorescent antibody (IFA) test and an A. platys species-specific ELISA were used as reference assays for the A. phagocytophilum and A. platys samples, respectively. The SNAP test demonstrated significantly higher sensitivity (84.7% for A. phagocytophilum and 83.1% for A. platys), compared to the VetScan test (39.0% for A. phagocytophilum and 57.6% for A. platys). The specificity of the SNAP test (95.8% for A. phagocytophilum and 99.2% for A. platys) was significantly greater than the VetScan test (85.6% for A. phagocytophilum and 82.5% for A. platys). In a separate clinic study, conducted within an A. phagocytophilum-endemic state (Minnesota) using 154 whole blood samples from client-owned dogs, the VetScan test was negative for 22 of 39 SNAP and IFA seropositive samples.

Analytical validation of a reference laboratory ELISA for the detection of feline leukemia virus p27 antigen.

Feline leukemia virus (FeLV) is an oncogenic retrovirus of cats. Immunoassays for the p27 core protein of FeLV aid in the detection of FeLV infections. Commercial microtiter-plate ELISAs have rapid protocols and visual result interpretation, limiting their usefulness in high-throughput situations. The purpose of our study was to validate the PetChek FeLV 15 ELISA, which is designed for the reference laboratory, and incorporates sequential, orthogonal screening and confirmatory protocols. A cutoff for the screening assay was established with 100% accuracy using 309 feline samples (244 negative, 65 positive) defined by the combined results of FeLV PCR and an independent reference p27 antigen ELISA. Precision of the screening assay was measured using a panel of 3 samples (negative, low-positive, and high-positive). The intra-assay coefficient of variation (CV) was 3.9-7.9%; the inter-assay CV was 6.0-8.6%. For the confirmatory assay, the intra-assay CV was 3.0-4.7%, and the inter-assay CV was 7.4-9.7%. The analytical sensitivity for p27 antigen was 3.7 ng/mL for inactivated whole FeLV and 1.2 ng/mL for purified recombinant FeLV p27. Analytical specificity was demonstrated based on the absence of cross-reactivity to related retroviruses. No interference was observed for samples containing added bilirubin, hemoglobin, or lipids. Based on these results, the new high-throughput design of the PetChek FeLV 15 ELISA makes it suitable for use in reference laboratory settings and maintains overall analytical performance.

Detection of Semivolatile Organic Compounds in Permeable Pavement Infiltrate.

The U.S. Environmental Protection Agency's Edison Environmental Center (EEC) has a research and demonstration permeable parking lot comprised of three different permeable systems: permeable asphalt, pervious concrete, and permeable interlocking concrete pavers. Water quality and quantity analysis has been ongoing since January 2010. This paper describes analysis of semivolatile organic compounds (SVOCs) to determine if hydrocarbons were in water that infiltrated through the permeable surfaces. SVOCs were analyzed in samples collected from 11 dates over a 3-year period, from February 8, 2010 to April 1, 2013. Results are broadly divided into three categories: 42 chemicals were never detected; 12 chemicals (11 chemical test) were detected at a rate of less than 10% or less; and 22 chemicals were detected at a frequency of 10% or greater (ranging from 10 to 66.5% detections). Fundamental and exploratory statistical analyses were performed on the 22 most observed chemicals. The statistical analyses were limited due to low frequency of detections and dilutions of samples, which impacted detection limits. The infiltrate data through three permeable surfaces were analyzed as nonparametric data by the Kaplan-Meier estimation method for fundamental statistics; there were some statistically observable differences in median concentration between pavement types when using Tarone-Ware comparison hypothesis test. A result was that three groups could be identified based on whether observed porous asphalt infiltrate concentration were greater than, similar to, or less than permeable interlocking concrete pavers infiltrate concentration. Identifying these three groups allowed one-way analysis on chemical attributes; the octonal water partitioning (logK OW), number of benzene rings, and molecular complexity were all significant. These 22 most observed chemicals in the infiltrate were further tested by Spearman rank order nonparametric for correlations between frequency of detection and chemical attributes; significant correlations were observed for porous asphalt frequency of detection and molecular weight (MW), Henry's constant, log K OW and molecular complexity, while both permeable concretes did not have any significant correlations between frequency of detection and chemical parameters. Conclusions from the statistical analyses on the 22 most frequently observed SVOCs in the infiltrate indicate that porous asphalt acts as a source for chemicals with low log K OW and MW and a sink for chemicals with high log K OW and MW, while no significant pattern was observed in the SVOC infiltrate concentrations of the two types of concrete.

Anemia and undernutrition among children aged 6-23 months in two agroecological zones of rural Ethiopia.

BACKGROUND: Child malnutrition during the first 1,000 days, commencing at conception, can have lifetime consequences. This study assesses the prevalence of anemia and undernutrition among children aged 6-23 months in midland and lowland agroecological zones of rural Ethiopia. METHODS: Cross-sectional data examining sociodemographic, anthropometry, hemoglobin levels, and meal frequency indicators were collected from 216 children aged 6-23 months and their mothers randomly selected from eight rural kebele (villages). RESULTS: Of 216 children, 53.7% were anemic, and 39.8%, 26.9%, and 11.6% were stunted, underweight, and wasted, respectively. The prevalence of anemia was higher in the lowland agroecological zone (59.5%) than the midland (47.6%). Among those children who were stunted, underweight, and wasted, 63.5%, 66.7%, and 68.0% were anemic, respectively. Child anemia was significantly associated with the child not achieving minimum meal frequency, sickness during the last 2 weeks before the survey, stunting and low body mass index, and with maternal hemoglobin and handwashing behavior. The prevalence of stunting was higher in the lowland agroecological zone (42.3%) than the midland (36.2%). The predictors of stunting were age and sex of the child, not achieving MMF, maternal body mass index, and age. As maternal height increases, the length for age of the children increases (P=0.003). CONCLUSION: The overall prevalence of anemia and undernutrition among children aged 6-23 months in these study areas is very high. The prevalence was higher in the lowland agro-ecological zone. Health information strategies focusing on both maternal and children nutrition could be sensible approaches to minimize stunting and anemia.

Seasonal variation in nutritional status and anemia among lactating mothers in two agro-ecological zones of rural Ethiopia: A longitudinal study.

OBJECTIVE: The aim of this study was to determine seasonal and agro-ecological variations in nutritional status, anemia, and associated factors among lactating women in rural Ethiopia. METHODS: We conducted a longitudinal study with 216 mothers in pre- and postharvest seasons in two agro-ecological zones of rural Ethiopia. We conducted interviews using a structured questionnaire, anthropometric measurements, and blood tests for anemia. We used multivariable linear regression models to identify independent predictors. RESULTS: The prevalence of anemia increased from postharvest (21.8%) to preharvest seasons (40.9%). Increases were from 8.6% to 34.4% in midland and from 34.2% to 46.3% in lowland agro-ecological zones. Of the mothers, 15% were anemic during both seasons. The prevalence of undernutrition, assessed using body mass index (BMI) < 18.5 kg/m(2), increased from 41.7% to 54.7% between the two seasons. Prevalence of maternal mid upper arm circumferences <22 cm also increased from 43.1% to 55.2% during the preharvest season. The seasonal effect was generally more pronounced in the midland community for all forms of malnutrition. Predictors of anemia were high parity of mother and low dietary diversity. Parity, number of children under the age of 5 y, and regional variation were predictors of low BMI among lactating mothers. CONCLUSION: The magnitude of malnutrition and anemia was significantly influenced by variations in season and agro-ecological zones. Interventions focused on agro-ecology and seasonal variation should be considered in addition to current strategies to alleviate malnutrition in lactating mothers.

SNAP Assay Technology.

The most widely used immunoassay configuration is the enzyme-linked immunosorbent assay (ELISA) because the procedure produces highly sensitive and specific results and generally is easy to use. By definition, ELISAs are immunoassays used to detect a substance (typically an antigen or antibody) in which an enzyme is attached (conjugated) to one of the reactants and an enzymatic reaction is used to amplify the signal if the substance is present. Optimized ELISAs include several steps that are performed in sequence using a defined protocol that typically includes application of sample and an enzyme-conjugated antibody or antigen to an immobilized reagent, followed by wash and enzyme reaction steps. The SNAP assay is an in-clinic device that performs each of the ELISA steps in a timed sequential fashion with little consumer interface. The components and mechanical mechanism of the assay device are described. Detailed descriptions of features of the assay, which minimize nonspecific binding and enhance the ability to read results from weak-positive samples, are given. Basic principles used in assays with fundamentally different reaction mechanisms, namely, antigen-detection, antibody-detection, and competitive assays are given. Applications of ELISA technology, which led to the development of several multianalyte SNAP tests capable of testing for up to 6 analytes using a single-sample and a single-SNAP device are described.

Antibodies reactive to Ehrlichia spp. are common in Oklahoma horses.

Abstract Tick infestations and infection with tick-borne agents are commonly recognized in horses in North America, but equine infection with true Ehrlichia spp. has not been described. To determine the degree to which horses in the south-central United States are naturally exposed to and infected with tick-borne disease agents, serum samples were collected at random (n=240) or from horses with active tick infestations (n=73) and tested by immunofluorescence antibody assay (IFA) and/or enzyme-linked immunosorbent assay (ELISA) for evidence of antibodies reactive to Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Positive samples were further evaluated by species-specific serology for antibodies reactive to E. canis and E. chaffeensis, and whole blood samples were tested by PCR for evidence of infection with E. canis, E. chaffeensis, E. ewingii, and an E. ruminantium-like organism referred to as the Panola Mountain Ehrlichia. Antibodies reactive to Ehrlichia spp. were identified in 8.75% (21/240) of the randomly acquired samples and 24.7% (18/73) of the serum samples from tick-infested horses, but species-specific ELISA and PCR failed to confirm exposure to or infection with any known Ehrlichia spp. Antibodies to Anaplasma spp. (5/313; 1.6%) and B. burgdorferi (3/313; 1.0%) were uncommon. These data suggest that horses in the south-central United States are likely exposed to a novel Ehrlichia sp. Further research is needed to identify the etiologic agent responsible for the serologic activity seen and to determine the clinical significance, if any, of this finding.

Trace metal accumulation in sediments and benthic macroinvertebrates before and after maintenance of a constructed wetland.

Stormwater best management practices (BMPs) require regular maintenance. The impact on trace metal concentrations in a constructed stormwater wetland BMP on Staten Island, New York, was investigated by analyzing sediment concentrations and tissue residues of the dominant macroinvertebrates (Tubifex tubifex) prior and subsequent to maintenance. Trace metal concentrations were assessed using standard serial extraction (for sediment) and acid digestion (for tissue burdens) techniques, followed by quantitative determination using graphite furnace atomic absorption spectrometry and inductively coupled plasma optical emission spectrometry, respectively. The results suggest that disturbance of sediment during maintenance of the BMP resulted in an increase in the most mobile fraction of trace metals, especially those associated with finer grained sediments (< 63 tm), and as a consequence, measured metal concentrations in macroinvertebrates increased. Regressions of a subset of metal concentrations (copper, lead, and zinc) in sediment and the macroinvertebrate tissue burden samples generally increased as a result of maintenance. A follow-up sampling event 9 months after maintenance demonstrated that the most readily available form of trace metal in the BMP was reduced, which supports (1) long-term sequestration of metals in the BMP and (2) that elevated bioavailability following maintenance was potentially a transient feature of the disturbance. This study suggests that in the long-term, performing sediment removal might help reduce bioavailability of trace metal concentrations in both the BMP and the receiving water to which a BMP discharges. However, alternative practices might need to be implemented to reduce trace metal bioavailability in the short-term.

Experimental primary and secondary infections of domestic dogs with Ehrlichia ewingii.

In this study, the infection dynamics of Ehrlichia ewingii, causative agent of granulocytotropic ehrlichiosis in dogs and humans, was examined in experimentally infected dogs by using a combination of physical examination, hematologic and biochemical analyses, and molecular and serologic assays. For the experimental trials, blood from an E. ewingii-infected dog was inoculated intravenously into two naïve dogs and two dogs with prior experimental exposure to E. ewingii (both were negative for E. ewingii DNA by polymerase chain reaction (PCR) assay, but seropositive from initial infection 8 and 10 months prior to challenge). A negative control dog was inoculated with blood from a negative dog. The two primary infection dogs were positive for E. ewingii DNA on DPI 4, remained consistently positive until DPI 60, and were intermittently positive until the end of the study (DPI 144). The two primary infection dogs developed antibodies reactive to E. ewingii by DPI 28 and remained seropositive for the duration of the study. Primary infected dogs had intermittent fever, thrombocytopenia, and leukopenia and some dogs were hyperphosphatemic and/or had elevated ALP levels. The two challenge dogs were positive for E. ewingii DNA on DPI 4 and 18, which was similar to the primary infection dogs, but the duration of E. ewingii DNA detection was shorter. Also, the two challenged dogs did not develop pyrexia or show any hematologic or biochemical abnormalities. E. ewingii was successfully transmitted between dogs by Amblyomma americanum, but not Rhipicephalus sanguineus. This study provides data on the infection dynamics of E. ewingii in dogs during primary and challenge infections and suggests that prior exposure may lessen clinical disease during subsequent infections.

Evaluation of peptide- and recombinant protein-based assays for detection of anti-Ehrlichia ewingii antibodies in experimentally and naturally infected dogs. [corrected]

OBJECTIVE: To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs. SAMPLE POPULATION: Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri. PROCEDURES: The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28) RESULTS: A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.

Ehrlichia ewingii infection and exposure rates in dogs from the southcentral United States.

We used PCR and a novel serologic assay to determine infection and exposure rates to Ehrlichia ewingii in dogs from an area of northeast Oklahoma and northwest Arkansas where Amblyomma americanum ticks are abundant. Of 143 dogs assayed, 13 (9.1%) harbored E. ewingii by PCR and 64 (44.8%) had antibodies to E. ewingii detected using a peptide-based microtiter plate ELISA. Dogs were more likely (P=0.001) to be positive by PCR if sampled in August (30.8%) but no association was found between seropositive status and month of collection of sample (P>0.05). Additional testing revealed PCR evidence of Ehrlichia chaffeensis (4/143; 2.8%) and Anaplasma platys (5/143; 3.5%) as well as antibodies reactive to E. chaffeensis (25/143; 17.5%), Ehrlichia canis (2/143; 1.4%), and Anaplasma spp. (8/143; 5.6%). Testing of another 200 dogs from the area revealed additional PCR and/or serologic evidence of E. ewingii, E. canis, E. chaffeensis, and A. platys. None of the 343 dogs evaluated had evidence of Borrelia burgdorferi exposure. These data support the interpretation that E. ewingii may be the primary agent of canine ehrlichiosis in this region, and suggest that diagnostic evaluation of dogs suspected to have a tick-borne disease should include assays targeting this organism.

Distribution of antibodies reactive to Borrelia lonestari and Borrelia burgdorferi in white-tailed deer (Odocoileus virginianus) populations in the eastern United States.

Southern tick-associated rash illness is a Lyme-like syndrome that occurs in the southern states. Borrelia lonestari, which has been suggested as a possible causative agent of southern tick-associated rash illness, naturally infects white-tailed deer (WTD; Odocoileus virginianus) and is transmitted by the lone star tick (Amblyomma americanum). To better understand the prevalence and distribution of Borrelia exposure among WTD, we tested WTD from 21 eastern states for antibodies reactive to B. lonestari using an indirect immunofluorescent antibody assay and Borrelia burgdorferi using the IDEXX SNAP 4Dx test. A total of 107/714 (15%) had antibodies reactive to B. lonestari, and prevalence of antibodies was higher in deer from southern states (17.5%) than in deer from northern states (9.2%). Using the SNAP 4DX test, we found that 73/723 (10%) were positive for B. burgdorferi, and significantly more northern deer (23.9%) were positive compared with southern deer (3.8%). Our data demonstrate that WTD are exposed to both Borrelia species, but antibody prevalence for exposure to the two species differs regionally and distributions correlate with the presence of Ixodes scapularis and A. americanum ticks.

Borrelia burgdorferi sensu lato species in Europe induce diverse immune responses against C6 peptides in infected mice.

The diversity of Lyme-borreliosis-inducing Borrelia species in Europe set high standards for the use of serodiagnostic test systems in terms of specificity and sensitivity. In the United States, the one-step C6 antibody test system based on the invariable domain IR6 of the VlsE molecule has been established as a successful diagnostic tool for testing canine samples. However, only a limited set of data are available regarding the antigenicity of the C6 peptides in an experimental murine model and sensitivity of the test regarding European Borrelia species. In order to investigate antibody reactions induced by these spirochetes, a total of 142 C3H/HeN mice were inoculated with Borrelia burgdorferi sensu stricto N40, B. garinii PBi, two isolates of B. afzelii, B. spielmanii A14S, B. valaisiana Rio6, B. valaisiana VS116, or B. lusitaniae. Infection of the mice was documented utilizing tissue culture and PCR. The IR6 sequences of B. burgdorferi sensu stricto B31, B. garinii IP90, and two B. afzelii ACAI strains have been used to synthesize and test additional C6 peptides. Compared to the well-established two-tiered test system, the results indicate that single C6 peptides derived from B. burgdorferi sensu stricto and B. garinii can be used in an enzyme-linked immunosorbent assay-based technique to detect murine antibodies induced by either agent. Little is known about the prevalence or pathogenicity of the B. afzelii strains in mammalian hosts, but our experimental data indicate differences in the C6 peptide test sensitivity for the detection of antibodies induced by different strains or isolates of B. afzelii.