Role of zinc metalloprotease (ZMPSTE24) in porcine reproductive and respiratory syndrome virus (PRRSV) replication in vitro.

The transmembrane zinc metalloprotease ZMPSTE24 works in cooperation with interferon-induced transmembrane protein 3 (IFITM3) to restrict entry of several enveloped viruses. We investigated the role of ZMPSTE24 in porcine reproductive and respiratory syndrome virus (PRRSV) replication. ZMPSTE24 overexpression significantly reduced PRRSV replication in MARC-145 cells. Interestingly, knockdown of endogenous ZMPSTE24 did not significantly impact virus replication. There was no significant difference in the percentage of PRRSV-positive cells and viral RNA copies at 3 hours postinfection (hpi) between cells transfected with ZMPSTE24-FLAG and the vector control. Our results suggest that ZMPSTE24 overexpression may restrict PRRSV replication at a post-entry step.

Intraductal Drug Delivery to the Breast: Effect of Particle Size and Formulation on Breast Duct and Lymph Node Retention.

Drug delivery by direct intraductal administration can achieve high local drug concentration in the breast and minimize systemic levels. However, the clinical application of this approach for breast cancer treatment is limited by the rapid clearance of the drug from the ducts. With the goal of developing strategies to prolong drug retention in the breast, this study was focused on understanding the influence of particle size and formulation on breast duct and lymph node retention. Fluorescent-labeled polystyrene (PS) particles ranging in size from 100 to 1000 nm were used to study the influence of particle size. Polylactic acid-co-glycolic acid (PLGA) was used to develop and test formulations for intraductal delivery. Cy 5.5, a near-IR dye, was encapsulated in PLGA microparticles, nanoparticles, and the in situ gel to study the biodistribution in rats using an in vivo imager. PS microparticles (1 µm) showed longer retention in the duct compared to 100 and 500 nm nanoparticles. The ductal retention half-life was 5-fold higher for PS microparticles compared to the nanoparticles. On the other hand, the free dye was cleared from the breast within 6 h. PLGA nanoparticles sustained the release of Cy 5.5 for >4 days. Microparticles and gel showed a much slower release than nanoparticles. PLGA in situ gel and microparticles were retained in the breast for up to 4 days, while the nanoparticles were retained in the breast for 2 days. PLGA nanoparticles and microparticles drained to the axillary lymph node and were retained for up to 24 and 48 h, respectively, while the in situ gel and the free dye did not show any detectable fluorescence in the lymph nodes. Taken together, the results demonstrate the feasibility of prolonged retention in the breast duct and lymph node by optimal formulation design. The findings can serve as a framework to design formulations for localized treatment of breast cancer.

δ-Tocopherol Effect on Endocytosis and Its Combination with Enzyme Replacement Therapy for Lysosomal Disorders: A New Type of Drug Interaction?

Induction of lysosomal exocytosis alleviates lysosomal storage of undigested metabolites in cell models of lysosomal disorders (LDs). However, whether this strategy affects other vesicular compartments, e.g., those involved in endocytosis, is unknown. This is important both to predict side effects and to use this strategy in combination with therapies that require endocytosis for intracellular delivery, such as lysosomal enzyme replacement therapy (ERT). We investigated this using δ-tocopherol as a model previously shown to induce lysosomal exocytosis and cell models of type A Niemann-Pick disease, a LD characterized by acid sphingomyelinase (ASM) deficiency and sphingomyelin storage. δ-Tocopherol and derivative CF3-T reduced net accumulation of fluid phase, ligands, and polymer particles via phagocytic, caveolae-, clathrin-, and cell adhesion molecule (CAM)-mediated pathways, yet the latter route was less affected due to receptor overexpression. In agreement, δ-tocopherol lowered uptake of recombinant ASM by deficient cells (known to occur via the clathrin pathway) and via targeting intercellular adhesion molecule-1 (associated to the CAM pathway). However, the net enzyme activity delivered and lysosomal storage attenuation were greater via the latter route. Data suggest stimulation of exocytosis by tocopherols is not specific of lysosomes and affects endocytic cargo. However, this effect was transient and became unnoticeable several hours after tocopherol removal. Therefore, induction of exocytosis in combination with therapies requiring endocytic uptake, such as ERT, may represent a new type of drug interaction, yet this strategy could be valuable if properly timed for minimal interference.

Identification of surface polysaccharides in akinetes, heterocysts and vegetative cells of Anabaena cylindrica using fluorescein-labeled lectins.

In response to environmental changes, Anabaena cylindrica differentiate three cell types: vegetative cells for photosynthesis, heterocysts for nitrogen fixation, and akinetes for stress survival. Cell-surface polysaccharides play important roles in cyanobacterial ecophysiology. In this study, specific cell-surface sugars were discovered in heterocysts, akinetes and vegetative cells of A. cylindrica using 20 fluorescein-labeled lectins. Both N-acetylglucosamine-binding lectins WGA and succinylated WGA bound specifically to the vegetative cells. Akinetes bound to three mannose-binding lectins (LCA, PSA, and ConA), and one of the galactose-binding lectins (GSL-I). Heterocyst also bound to ConA. However, the heterocysts in all4388 mutant of Anabaena sp. PCC 7120, in which the putative polysaccharide export protein gene all4388 was disrupted, exhibited diminished binding to ConA. Identification of distinct cell-surface sugar helped us to understand the role of polysaccharide for each cell type. Fluorescence-activated cell sorting may be applicable in isolating each cell type for comparative "omics" studies among the three cell types.

Simultaneous gene inactivation and promoter reporting in cyanobacteria.

Determining spatiotemporal gene expression and analyzing knockout mutant phenotypes have become powerful tools in elucidating the function of genes; however, genetic approaches for simultaneously inactivating a gene and monitoring its expression have not been reported in the literature. In this study, we designed a dual-functional gene knockout vector pZR606 that contains a multiple cloning site (MCS) for inserting the internal fragment of a target gene, with a gfp gene as its transcriptional marker located immediately downstream of the MCS. By using this gene knockout system, we inactivated ava_2679 from Anabaena variabilis ATCC 29413, as well as all2508, alr2887, alr3608, and all4388 from Anabaena sp. strain PCC 7120. The ava_2679 knockout mutant fails to grow diazotrophically. Morphological analysis of ava_2679 knockout mutant after nitrogen step-down revealed defective junctions between heterocysts and adjacent vegetative cells, and the heterocyst was 1.53-fold longer compared to wild-type heterocysts. The alr2887, all4388, and alr3608 mutant colonies turned yellow and showed lack of protracted growth when deprived of fixed nitrogen, consistent with the previous reports that alr2887, all4388, and alr3608 are Fox genes. The all2508 encodes a GTP-binding elongation factor (EF4/LepA), and its knockout mutant exhibited reduced diazotrophic growth. The heterocyst development of all2508 knockout was significantly delayed, and only about 4.0 % of vegetative cells differentiated to heterocysts after nitrogen deprivation for 72 h, decreased 49.6 % compared to wild-type. Thus, we discovered that All2508 may regulate heterocyst development spatiotemporally. Concurrently, the GFP reporter revealed that all five target gene expressions were up-regulated in response to nitrogen deprivation. We demonstrated that the pZR606-based specific gene knockout approach worked effectively for the five selected genes, including four previously identified Fox genes or Fox gene homolog, and a previously unknown function of gene all2508. Thus, gene expression and phenotypic analysis of mutants can be achieved simultaneously by targeted gene inactivation using the pZR606-based system. This combined approach for targeted gene inactivation and its promoter reporting with GFP may be broadly applicable to the study of gene function in other prokaryotic organisms.

Silencing RNA-Mediated Knockdown of IFITM3 Enhances Senecavirus A Replication.

Senecavirus A (SVA) is a non-enveloped, positive sense, single-stranded RNA virus that causes vesicular diseases in pigs. Interferon-induced transmembrane 3 (IFITM3) is an interferon-stimulated gene (ISG) that exhibits broad antiviral activity. We investigated the role of IFITM3 in SVA replication. Both viral protein expression and supernatant virus titer were significantly increased when endogenous IFITM3 was knocked down by approximately 80% in human non-smallcell lung carcinoma cell line (NCI-H1299) compared to silencing RNA control. Interestingly, overexpression of exogenous IFITM3 in NCI-H1299 cells also significantly enhanced viral protein expression and virus titer compared to vector control, which was positively correlated with induction of autophagy mediated by IFITM3 overexpression. Overall, our results indicate an antiviral role of endogenous IFITM3 against SVA. The exact molecular mechanisms by which endogenous IFITM3 limits SVA replication remain to be determined in future studies.

Satellite Microwave Remote Sensing for Environmental Modeling of Mosquito Population Dynamics.

Environmental variability has important influences on mosquito life cycles and understanding the spatial and temporal patterns of mosquito populations is critical for mosquito control and vector-borne disease prevention. Meteorological data used for model-based predictions of mosquito abundance and life cycle dynamics are typically acquired from ground-based weather stations; however, data availability and completeness are often limited by sparse networks and resource availability. In contrast, environmental measurements from satellite remote sensing are more spatially continuous and can be retrieved automatically. This study compared environmental measurements from the NASA Advanced Microwave Scanning Radiometer on EOS (AMSR-E) and in situ weather station data to examine their ability to predict the abundance of two important mosquito species (Aedes vexans and Culex tarsalis) in Sioux Falls, South Dakota, USA from 2005 to 2010. The AMSR-E land parameters included daily surface water inundation fraction, surface air temperature, soil moisture, and microwave vegetation opacity. The AMSR-E derived models had better fits and higher forecasting accuracy than models based on weather station data despite the relatively coarse (25-km) spatial resolution of the satellite data. In the AMSR-E models, air temperature and surface water fraction were the best predictors of Aedes vexans, whereas air temperature and vegetation opacity were the best predictors of Cx. tarsalis abundance. The models were used to extrapolate spatial, seasonal, and interannual patterns of climatic suitability for mosquitoes across eastern South Dakota. Our findings demonstrate that environmental metrics derived from satellite passive microwave radiometry are suitable for predicting mosquito population dynamics and can potentially improve the effectiveness of mosquito-borne disease early warning systems.

Trichostrongyle infections in domestic ruminants from Egypt: A systematic review and meta-analysis.

Trichostrongylid nematodes can cause serious loss in the livestock economy; nevertheless, infections with these ubiquitous nematodes in animals from developing countries are largely neglected. The present paper provides a systematic review and meta-analysis to evaluate the status of trichostrongyle infections in domestic ruminants from Egypt. Out of 626 collected publications, 118 were defined suitable for inclusion in this review and represented trichostrongyle infections in 5 ruminant species (sheep, goats, cattle, buffaloes and camels) from Egypt. Published surveys have mostly focused on sheep, which had the highest (44.8%) pooled prevalence of trichostrongyle infections based on 95% confidence interval (35.9-53.6%) with no significant variations among sheep in different Egyptian regions. Goats had lower infection prevalence (31.2%, 21.5-40.8%) in comparison to sheep, and no significant regional differences were also found. The management and marketing practices likely account for the wide distribution of infection among small ruminants across Egypt. Variable trichostrongyle infection rates were estimated for camels (38.8%, 28.9-48.7%), cattle (27.4%, 14.4-40.3%) and buffaloes (12.2%, 8.0-16.4%). The prevalence of infection was significantly high during winter (52.1%, 32.2-72.1%), which provide favorable conditions for development and survival of larvae on pastures. The most common trichostrongyle identified in infected animals was Haemonchus contortus. The parasite was detected in 38.8% (29.9-47.6%) of infected sheep, 35.3% (24.8-45.7%) of infected goats and in 40.6% (18.6-62.5%) of infected camels. Clinical infections have been reported in a limited number of studies. Analysis of fecal egg counts (FECs) revealed that more than half (52.7%, 30.4-75.0%) of the tested sheep had low counts (<500 eggs per gram EPG), whereas only a few sheep (6.1%, 3.5-8.7%) had high counts >2000 EPG, implying that subclinical infections are common; however, the clinical infections cannot be completely ruled out. Anthelmintic resistance does not appear to be serious in trichostrongyle populations infecting ruminants from Egypt; nonetheless there have been a few cases of albendazole resistance in trichostrongyles infecting sheep. This paper contributes to a better understanding of the epidemiology and economic implications of trichostrongyle infections in ruminants from Egypt, which is crucial for establishing effective control strategies against these ubiquitous nematodes.

Integrated Forecasts Based on Public Health Surveillance and Meteorological Data Predict West Nile Virus in a High-Risk Region of North America.

BACKGROUND: West Nile virus (WNV), a global arbovirus, is the most prevalent mosquito-transmitted infection in the United States. Forecasts of WNV risk during the upcoming transmission season could provide the basis for targeted mosquito control and disease prevention efforts. We developed the Arbovirus Mapping and Prediction (ArboMAP) WNV forecasting system and used it in South Dakota from 2016 to 2019. This study reports a post hoc forecast validation and model comparison. OBJECTIVES: Our objective was to validate historical predictions of WNV cases with independent data that were not used for model calibration. We tested the hypothesis that predictive models based on mosquito surveillance data combined with meteorological variables were more accurate than models based on mosquito or meteorological data alone. METHODS: The ArboMAP system incorporated models that predicted the weekly probability of observing one or more human WNV cases in each county. We compared alternative models with different predictors including a) a baseline model based only on historical WNV cases, b) mosquito models based on seasonal patterns of infection rates, c) environmental models based on lagged meteorological variables, including temperature and vapor pressure deficit, d) combined models with mosquito infection rates and lagged meteorological variables, and e) ensembles of two or more combined models. During the WNV season, models were calibrated using data from previous years and weekly predictions were made using data from the current year. Forecasts were compared with observed cases to calculate the area under the receiver operating characteristic curve (AUC) and other metrics of spatial and temporal prediction error. RESULTS: Mosquito and environmental models outperformed the baseline model that included county-level averages and seasonal trends of WNV cases. Combined models were more accurate than models based only on meteorological or mosquito infection variables. The most accurate model was a simple ensemble mean of the two best combined models. Forecast accuracy increased rapidly from early June through early July and was stable thereafter, with a maximum AUC of 0.85. The model predictions captured the seasonal pattern of WNV as well as year-to-year variation in case numbers and the geographic pattern of cases. DISCUSSION: The predictions reached maximum accuracy early enough in the WNV season to allow public health responses before the peak of human cases in August. This early warning is necessary because other indicators of WNV risk, including early reports of human cases and mosquito abundance, are poor predictors of case numbers later in the season. https://doi.org/10.1289/EHP10287.

Effects of magnolol on UVB-induced skin cancer development in mice and its possible mechanism of action.

BACKGROUND: Magnolol, a plant lignan isolated from the bark and seed cones of Magnolia officinalis, has been shown to have chemopreventive effects on chemically-induced skin cancer development. The objectives of this investigation are to study the anticarcinogenic effects of magnolol on UVB-induced skin tumor development in SKH-1 mice, a model relevant to humans, and determine the possible role of apoptosis and cell cycle arrest involved in the skin tumor development. METHODS: UVB-induced skin carcinogenesis model in SKH-1 mice was used for determining the preventive effects of magnolol on skin cancer development. Western blottings and flow cytometric analysis were used to study the effects of magnolol on apoptosis and cell cycle. RESULTS: Magnolol pretreated groups (30, 60 µ g) before UVB treatments (30 mJ/cm2, 5 days/week) resulted in 27-55% reduction in tumor multiplicity as compared to control group in SKH-1 mice. Magnolol pretreatment increased the cleavage of caspase-8 and poly-(-ADP-ribose) polymerase (PARP), increased the expression of p21, a cell cycle inhibitor, and decreased the expression of proteins involved in the G2/M phase of cell cycle in skin samples from SKH-1 mice.Treatment of A431 cells with magnolol decreased cell viability and cell proliferation in a concentration dependent manner. Magnolol induced G2/M phase cell cycle arrest in A431 cells at 12 h with a decreased expression of cell cycle proteins such as cyclin B1, cyclin A, CDK4, Cdc2 and simultaneous increase in the expression of Cip/p21, a cyclin-dependent kinase inhibitor. Magnolol induced apoptosis in vivo and in vitro with an increased cleavage of caspase-8 and PARP. Phospho-signal transducers and activators of transcription 3 (Tyr705), B-Raf, p-MEK, and p-AKT were down-regulated, whereas phosphorylation of ERK was induced by magnolol in A431 cells. CONCLUSIONS: Magnolol pretreatments prevent UVB-induced skin cancer development by enhancing apoptosis, causing cell cycle arrest at G2/M phase, and affecting various signaling pathways. Magnolol could be a potentially safe and potent anticarcinogenic agent against skin cancer.

Structure-skin permeability relationship of dendrimers.

PURPOSE: To investigate skin penetration of poly (amidoamine) (PAMAM) dendrimers as a function of surface charge and molecular weight in presence and absence of iontophoresis. METHODS: Dendrimers were labeled with fluoroisothiocynate (FITC); skin penetration of dendrimers was studied using excised porcine skin in-vitro. Skin penetration of FITC-labeled dendrimers was quantified using confocal laser scanning microscope (CLSM). G2-G6 NH(2), G3.5-COOH and G4-OH dendrimers were used. RESULTS: Cationic dendrimers showed higher skin penetration than neutral and anionic dendrimers. Skin penetration of cationic dendrimer increased linearly with increase in treatment time. Iontophoresis enhanced skin penetration of cationic and neutral dendrimers. Increase in current strength and current duration increased skin transport of dendrimers. Passive and iontophoretic skin penetration of cationic dendrimers was inversely related to their molecular weight. Dendrimer penetrated the skin through intercellular lipids and hair follicles. With iontophoresis, dendrimer was also found in localized skin regions. CONCLUSIONS: The study demonstrates that the physicochemical properties of dendrimers influence their skin transport. Findings can be used to design dendrimer-based nanocarriers for drug delivery to skin.

Weather and land cover influences on mosquito populations in Sioux Falls, South Dakota.

This study compared the spatial and temporal patterns of Culex tarsalis Coquillett and Aedes vexans Meigen populations and examined their relationships with land cover types and climatic variability in Sioux Falls, SD. Between 24 and 30 CDC CO2-baited light traps were set annually in Sioux Falls from May to September 2005-2008. Land cover data were acquired from the 2001 National Land Cover Dataset and the percentages of selected land cover types were calculated within a 600-m buffer zone around each trap. Meteorological information was summarized from local weather stations. Cx. tarsalis exhibited stronger spatial autocorrelation than Ae. vexans. Land cover analysis indicated that Cx. tarsalis was positively correlated with grass/hay, and Ae. vexans was positively correlated with wetlands. No associations were identified between irrigation and the host-seeking population of each species. Higher temperature in the current week and 2 wk prior and higher precipitation 3-4 wk before collection of host-seeking adult mosquitoes had positive influences on Cx. tarsalis abundance. Temperature in the current week and rainfall 2-3 wk before sampling had positive influences on Ae. vexans abundance. This study revealed the different influences of weather and land cover on important mosquito species in the Northern Great Plains region, which can be used to improve local vector control strategies and West Nile virus prevention efforts.

Triple lectin staining of trichostrongyle eggs from naturally infected small ruminants.

Trichostrongyle nematodes can be a major threat to the profitability of small ruminant producers depending of the species and intensity of trichostrongyles parasitizing their herd. Haemonchus contortus, Teladorsagia circumcincta, and Trichostrongylus colubriformis are typically the most common and clinically important species. Three lectins (PNA, LCA and AAL) have been reported to bind specifically to eggs from these three genera and therefore could be used to quantify the intensity of each species in individual animals. Peanut agglutinin (PNA) has been the most commonly tested lectin because it selectively binds intensely to eggs of the most pathogenic species, H. contortus. Lens culinaris agglutinin (LCA) and Aleuria aurantia agglutinin (AAL) have shown specificity to T. circumcincta and Trichostrongylus spp. respectively, however, these lectins have only been evaluated using eggs harvested directly from adult females, and not from fecal samples. The purpose of the present study is to describe a method to sequentially stain H. contortus, T. circumcincta and Trichostrongylus spp. fecal eggs with PNA, LCA and AAL, and then evaluate the resultant staining patterns seen with eggs collected from a naturally infected goat shown with PCR to contain H. contortus, T. circumcincta, Ostertagia leptospicularis, Trichostrongylus colubriformis and Trichostrongylus axei eggs. These results were also compared with patterns observed with eggs stained with single lectins and double combinations of lectins. The various patterns were then compared to those seen with egg samples collected from an ewe shown to only contain H. contortus. PNA bound intensely and uniformly to all eggs from samples containing only H. contortus eggs; however, some eggs additionally bound LCA and AAL in localized patches of varying size, and a few eggs exhibited intense and uniform binding of all three lectins. Single PNA-staining of goat samples containing the five trichostrongyles species identified most eggs as H. contortus, and triple-staining showed patterns consistent with those seen for H. contortus. Binding of AAL to non-Haemonchus eggs was uniform but showed significant variations in intensity. Lesser staining eggs tended to also stain intensely with LCA, which is consistent with published binding pattern for T. circumcincta. Most eggs that AAL bound intensely to did not bind with LCA, which is consistent with published binding pattern for Trichostrongylus spp. Autofluorescence was observed with the DAPI filter-cube among most non-Haemonchus eggs. This study demonstrates the need for additional field studies to further validate the specificity of these three lectins for use in identifying eggs from the three species of trichostrongyles.

Combined Molecular and Lectin Binding Assays to Identify Different Trichostrongyle Eggs in Feces of Sheep and Goats from Egypt.

BACKGROUND: Trichostrongyles are common causes of parasitic gastroenteritis in sheep and goats worldwide. Accurate identification of these nematodes to the genus and/or species level is important for therapy selection and control strategies. In the present study, molecular and egg-lectin binding approaches were employed to identify the most economically important trichostrongyles circulating in sheep and goat herds from six districts in Dakahlia governorate, Egypt. MATERIALS: Fecal samples from 653 and 205 goats reared within 17 herds were collected and tested for the trichostrongyle eggs using the modified Wisconsin sucrose flotation method. For identification of the trichostrongyle(s) present, eggs from 75 (63 sheep and 12 goats) samples which had high egg count (EPG) and pooled eggs (n = 19 pools, 15 sheep and 4 goats) from samples with moderate or low EPGs were examined. Molecular examination was conducted amplifying the ITS2 region of the rDNA for six different trichostrongyles in individual PCR reactions. For egg-lectin bindings, 4 fluorescently-labeled specific lectins were used; peanut agglutinin (PNA) for Haemonchus contortus, Aleuria aurantia agglutinin (AAL) for Trichostrongylus species, Lens culinaris agglutinin (LCA) for Teladorsagia circumcnicta and Lotus tetragonolobus lectin (LTL) for Cooperia species. RESULTS: Fourteen (82.3%) herds were found infected, of which trichostrongyle eggs were detected in fecal samples of 26.5% (173/653) of sheep and 10.2% (21/205) of goats. Results of the PCR and lectin bindings were compatible and 4 trichostrongyles were detected: H. contortus, T. circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Haemonchus contortus eggs were found in all the infected herds, and as the single species in 21 and 5 of sheep and goat samples, respectively. Lectin stained smears demonstrated the dominance of H. contortus eggs over eggs of the other detected trichostrongyles. Eleven herds were found infected with T. axei as the second most prevalent trichostrongyle; however, few AAL-stained eggs were noticed in the positive samples. Mixed infections were frequently detected as H. contortus-T. axei combination. Infections with T. circumcincta were noted in sheep samples from two herds, but not in any sample from the goats. No Ostertagia leptospicularis, Cooperia curticei or Nematodirus species were noted among the tested samples. CONCLUSIONS: This is the first molecular and lectin binding survey to determine the species composition of trichostrongyles infecting sheep and goats from Egypt. Haemonchus contortus plays the principal role in small ruminant trichostrongylosis in Egypt. Egg-lectin staining shows promise for future for its application in routine diagnosis as a rapid and simple technique. Findings of the earlier reports from Egypt are tabulated and reviewed.

Epidemiology and Control of Gastrointestinal Nematodes of Cattle in Northern Climates.

Parasite species infecting cattle throughout northern North American are generally the same as those found throughout North America. Throughout Canada, cattle are primarily infected with Ostertagia ostertagi and Cooperia oncophora, whose larvae survive cold winters within soil of pastures. Overwintering larvae of these species maintain a temporary population of refugia available in spring to grazing cattle. Cattle from northern United States are also infected with Cooperia punctata and Haemonchus placei, whose larvae cannot survive cold winters within pastures. Anthelmintics with persistent activity are used during spring to recover some of these losses; however, anthelmintic resistance limits effectiveness of this strategy.

Epidemic West Nile Virus Infection Rates and Endemic Population Dynamics Among South Dakota Mosquitoes: A 15-yr Study from the United States Northern Great Plains.

Mosquito surveillance has been conducted across South Dakota (SD) to record and track potential West Nile virus (WNV) vectors since 2004. During this time, communities from 29 counties collected nearly 5.5 million mosquitoes, providing data from over 60,000 unique trapping nights. The nuisance mosquito, Aedes vexans (Meigen) was the most abundant species in the state (39.9%), and most abundant in most regions. The WNV vector, Culex tarsalis Coquillett (Diptera: Culicidae), was the second most abundant species (20.5%), and 26 times more abundant than the other Culex species that also transmit WNV. However, geographic variation did exist between WNV vector species, as well as relative abundance of vector and nuisance mosquitoes. The abundance of Ae. vexans decreased from east to west in South Dakota, resulting in an increase in the relative abundance of Cx. tarsalis. Other species are reported in this study, with various relative abundances throughout the different regions of South Dakota. WNV infection rates of mosquitoes showed that Cx. tarsalis had the most positive sampling pools and the highest vector index of all the species tested. This study addressed the need for an updated summary of the predominant mosquito species present in the United States Northern Great Plain and provides infection rate data for WNV among these predominant species.

Chemopreventive effects of sarcophine-diol on ultraviolet B-induced skin tumor development in SKH-1 hairless mice.

Sarcophine-diol (SD), one of the structural modifications of sarcophine, has shown chemopreventive effects on 12-dimethylbenz(a)anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor development in female CD-1 mice. The objective of this study was to determine the chemopreventive effects of SD on UVB-induced skin tumor development in hairless SKH-1 mice, a model more relevant to human skin cancer, and to determine the possible mechanisms of action. Carcinogenesis was initiated and promoted by UVB radiation. Female hairless SKH-1 mice were divided into two groups having 27 mice in each group: control and SD treatment. The control group was topically treated with 100 microL acetone and SD treatment group was topically treated with SD (30 microg/100 microL in acetone) 1 hour before each UVB radiation for 32 weeks. Tumor counts were recorded on a weekly basis for 30 weeks. Effects of SD on the expression of caspases were investigated to elucidate the possible mechanism of action. The proteins from epidermal homogenates of experimental mice were used for SDS-PAGE and Western blotting using specific antibodies against caspase-3, caspase-8 and caspase-9 respectively. TUNEL assay was used for determining DNA fragmented apoptotic cells in situ. Results showed that at the end of experiment, tumor multiplicity in control and SD treatment groups was 25.8 and 16.5 tumors per mouse respectively. Furthermore, Topical treatment of SD induced DNA fragmented apoptotic cells by upgrading the expressions of cleaved caspase-3 and caspase-8. This study clearly suggested that SD could be an effective chemopreventive agent for UVB-induced skin cancer by inducing caspase dependent apoptosis.

Egg autofluorescence and options for detecting peanut agglutinin binding for the identification of Haemonchus contortus eggs in fecal samples.

Quantifying eggs from Haemonchus and other trichostrongyle genera in sheep and goat fecal samples is important for evaluating control and treatment strategies for this family of nematodes with divergent pathologies, capabilities for anthelmintic resistance and environmental susceptibilities. Unfortunately, egg morphology among most of the genera do not differ enough to support the accurate identification of these genera with standard microscopic techniques. Several studies have identified specific lectins which bind selectively to sugars located on the egg surfaces for individual genera among the trichostrongyles. To detect lectins binding to these eggs, they must be directly or indirectly bound to fluorophores, and observed with an epi-fluorescence microscope. The binding of multiple lectins to isolated eggs from a fecal sample can be simultaneously detected if fluorophores are used whose excitation and emission spectra do not overlap, and this would enable the development of a fluorescence-based diagnostic test that identifies multiple trichostrongyle genera within each sample. The present study compared the usefulness of different, commercially available detection systems for use in detecting lectin binding to trichostrongyle eggs. Comparisons were made using the detection of PNA binding to H. contortus eggs with the goal of finding three systems with color spectra that do not overlap. These evaluations included both fluorophores directly conjugated to PNA in a one-step incubation protocol and a two-step incubation protocol involving biotinylated PNA and streptavidin conjugated to different fluorophores. Autofluorescence can affect the efficiency of any fluorescence-based detection system, and significant autofluorescence was observed among the unstained H. contortus eggs with the DAPI-type fluorescence filter, but it was significantly lower with the FITC-type filter and was virtually absent with the rhodamine-type filter. This study demonstrated that all the PNA detection methods tested with H. contortus eggs generated fluorescence intensities (FIs) that were significantly above the autofluorescence generated by the eggs among the three different fluorescence filters. Fluorescence intensities from PNA directly conjugated to either the FITC or rhodamine fluorophores were not different, but the lower autofluoresence in the rhodamine-type filter will enable this fluorophore to be detected more efficiently. Use of biotinylated PNA combined with streptavidin-conjugated to synthetic fluorophores (Alexa Fluor 405, 488 and 546) significantly increased FIs over that of the directly conjugated PNA, but there were no significant differences in FIs among these three biotin-avidin conjugation fluorophores. This biotin-avidin system required two incubation steps. Doubling the concentration of PNA also provided increased FI, at least for the biotin-avidin system. Adding an additional amplification step to the biotin-avidin system involving biotinylated anti-streptavidin followed by the streptavidin-Alexa Fluor complex also provided additional fluorescence.

Development and biochemical and immunological characterization of early passage and immortalized bovine intestinal epithelial cell lines from the ileum of a young calf.

The intestinal epithelium is a major site of interaction with pathogens. In bovine intestinal epithelial cells (BIECs), Toll-like receptors (TLRs) play an important role in innate immune responses against enteric pathogens. This study is aimed at establishing a stable bovine intestinal epithelial cell line that can be maintained by a continuous passage so that studies on innate immune responses against various enteric pathogens can be performed. The main goal was to establish pure cultures of primary and immortalized bovine intestinal epithelial cells from the ileum and then characterize them biochemically and immunologically. Mixed epithelial and fibroblast bovine ileal intestinal cultures were first established from a 2-day old calf. Limiting dilution method was used to obtain a clone of epithelial cells which was characterized using immunocytochemistry (ICC). The selected clone BIEC-c4 was cytokeratin positive and expressed low levels of vimentin, confirming the epithelial cell phenotype. Early passage BIEC-c4 cells were transfected with either simian virus 40 (SV40) large T antigen or human telomerase reverse transcriptase (hTERT), or human papillomavirus (HPV) type 16E6/E7 genes to establish three immortalized BIEC cell lines. The expression of SV40, hTERT and HPV E6/E7 genes in immortalized BIECs was confirmed by a polymerase chain reaction (PCR). Immunocytochemistry and immunofluorescence assays also confirmed the expression of SV40, hTERT and HPV E6 proteins. The immortalized BIECs were cytokeratin positive and all except HPV-BIECs expressed low levels of vimentin. A growth kinetics study indicated that there were no significant differences in the doubling time of immortalized BIECs as compared to early passage BIEC-c4 cells. All four BIEC types expressed TLR 1-10 genes, with TLR 3 and 4 showing higher expression across all cell types. These newly established early passage and immortalized BIEC cell lines should serve as a good model for studying infectivity, pathogenesis and innate immune responses against enteric pathogens.

First Evidence of Teladorsagia circumcincta Infection in Sheep from Egypt.

Trichostrongylid nematodes are a common cause of gastroenteritis in sheep. Despite its worldwide distribution, Teladorsagia circumcincta has not been included in reports listing the various trichostrongyles infecting sheep from Egypt. Herein, we describe the presence of 2 T. circumcincta haplotypes infecting small ruminants from Egypt. For this study, fresh fecal samples were collected from 340 sheep and 115 goats reared at 5 districts in Dakahlia governorate and its surroundings, Egypt. Trichostrongyle eggs were harvested from the samples, and then subjected to DNA isolation and analysis. Polymerase chain reaction (PCR) amplification was carried out for the second internal transcribed spacer of ribosomal DNA (ITS2 rDNA). Purified PCR products of T. circumcincta were sequenced, and the revealed sequences were subjected to the nucleotide and phylogenetic analysis. A relatively high prevalence of trichostrongyles eggs was identified in sheep (33.2%) and a lower prevalence was found in goats (14.7%). Molecular analysis revealed, for the first time, 2 sheep herds from Egypt that were infected with T. circumcincta. Both infected herds were raised by the Bedouins in rural areas of El Mahalla El Kubra city. No T. circumcincta infections were found in any of the goats. Nucleotide sequence analysis revealed 2 haplotypes (Te1 and Te2) from 7 successfully sequenced samples (5 from the first and 2 from the second herd). Te1 was the major haplotype in both herds, and Te2 was retrieved from a single sample. Phylogenetic analysis displayed that the Te1 haplotype clustered with one from Cyprus, which might have been introduced to Egypt via goats imported from Cyprus due to a program to improve meat and milk production in Egypt. The present results could be beneficial in understanding the epidemiology of T. circumcincta and other trichostrongyles in Egypt, and have implementations in the effective control strategies used in this region.