Cytostatic Effects of Polyethyleneimine Surfaces on the Mesenchymal Stromal Cell Cycle.

Polyelectrolytes assembled layer-by-layer (PEMs) are commonly used as functional coatings to build-up biological interfaces, particularly suitable as compatible layers for the interaction with a biological medium, providing suitable conditions to promote or prevent cell seeding while maintaining the phenotype. The proper assessment of the biocompatibility of PEMs and the elucidation of the related mechanisms are therefore of paramount importance. In this study, we report in detail the effect of two different PEM endings, polystyrene sulfonate (PSS) and polyethylenimine (PEI), respectively, on the cell adhesion, growth, and viability of human bone mesenchymal stromal cells (MSCs). The results have shown that PSS-ended substrates appear to be the most suitable to drive the cell adhesion and phenotype maintenance of MSCs, showing good biocompatibility. On the contrary, while the cells seem to adhere more quickly and strongly on the PEI-ended surfaces, the interaction with PEI significantly affects the growth and viability, reducing the cell spreading capability, by sequestering the adhesion molecules already in the very early steps of cell-substrate contact. These results point to the promotion of a cytostatic effect of PEI, rather than the often-claimed cytotoxicity.

Corrigendum: Monitoring the Spread of Swine Enteric Coronavirus Diseases in the United States in the Absence of a Regulatory Framework.

[This corrects the article DOI: 10.3389/fvets.2016.00018.].

Time-series analysis for porcine reproductive and respiratory syndrome in the United States.

Industry-driven voluntary disease control programs for swine diseases emerged in North America in the early 2000's, and, since then, those programs have been used for monitoring diseases of economic importance to swine producers. One example of such initiatives is Dr. Morrison's Swine Health Monitoring Project, a nation-wide monitoring program for swine diseases including the porcine reproductive and respiratory syndrome (PRRS). PRRS has been extensively reported as a seasonal disease in the U.S., with predictable peaks that start in fall and are extended through the winter season. However, formal time series analysis stratified by geographic region has never been conducted for this important disease across the U.S. The main objective of this study was to use approximately seven years of PRRS incidence data in breeding swine herds to conduct time-series analysis in order to describe the temporal patterns of PRRS outbreaks at the farm level for five major swine-producing states across the U.S. including the states of Minnesota, Iowa, North Carolina, Nebraska and Illinois. Data was aggregated retrospectively at the week level for the number of herds containing animals actively shedding PRRS virus. Basic descriptive statistics were conducted followed by autoregressive integrated moving average (ARIMA) modelling, conducted separately for each of the above-mentioned states. Results showed that there was a difference in the nature of PRRS seasonality among states. Of note, when comparing states, the typical seasonal pattern previously described for PRRS could only be detected for farms located in the states of Minnesota, North Carolina and Nebraska. For the other two states, seasonal peaks every six months were detected within a year. In conclusion, we showed that epidemic patterns are not homogeneous across the U.S, with major peaks of disease occurring through the year. These findings highlight the importance of coordinating alternative control strategies in different regions considering the prevailing epidemiological patterns.

OptisampleTM: Open web-based application to optimize sampling strategies for active surveillance activities at the herd level illustrated using Porcine Respiratory Reproductive Syndrome (PRRS).

Porcine reproductive and respiratory syndrome virus (PRRSv) infection causes a devastating economic impact to the swine industry. Active surveillance is routinely conducted in many swine herds to demonstrate freedom from PRRSv infection. The design of efficient active surveillance sampling schemes is challenging because optimum surveillance strategies may differ depending on infection status, herd structure, management, or resources for conducting sampling. Here, we present an open web-based application, named 'OptisampleTM', designed to optimize herd sampling strategies to substantiate freedom of infection considering also costs of testing. In addition to herd size, expected prevalence, test sensitivity, and desired level of confidence, the model takes into account the presumed risk of pathogen introduction between samples, the structure of the herd, and the process to select the samples over time. We illustrate the functionality and capacity of 'OptisampleTM' through its application to active surveillance of PRRSv in hypothetical swine herds under disparate epidemiological situations. Diverse sampling schemes were simulated and compared for each herd to identify effective strategies at low costs. The model results show that to demonstrate freedom from disease, it is important to consider both the epidemiological situation of the herd and the sample selected. The approach illustrated here for PRRSv may be easily extended to other animal disease surveillance systems using the web-based application available at http://stemma.ahc.umn.edu/optisample.

Assessment of air sampling methods and size distribution of virus-laden aerosols in outbreaks in swine and poultry farms.

Swine and poultry viruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), porcine epidemic diarrhea virus (PEDV), and highly pathogenic avian influenza virus (HPAIV), are economically important pathogens that can spread via aerosols. The reliability of methods for quantifying particle-associated viruses as well as the size distribution of aerosolized particles bearing these viruses under field conditions are not well documented. We compared the performance of 2 size-differentiating air samplers in disease outbreaks that occurred in swine and poultry facilities. Both air samplers allowed quantification of particles by size, and measured concentrations of PRRSV, PEDV, and HPAIV stratified by particle size both within and outside swine and poultry facilities. All 3 viruses were detectable in association with aerosolized particles. Proportions of positive sampling events were 69% for PEDV, 61% for HPAIV, and 8% for PRRSV. The highest virus concentrations were found with PEDV, followed by HPAIV and PRRSV. Both air collectors performed equally for the detection of total virus concentration. For all 3 viruses, higher numbers of RNA copies were associated with larger particles; however, a bimodal distribution of particles was observed in the case of PEDV and HPAIV.

Six-year surveillance of Newcastle disease virus in wild birds in north-eastern Spain (Catalonia).

Given that Newcastle disease (ND) is one of the major threats for the poultry industry, testing of Newcastle disease virus (NDV) has been carried out since 2010 in cases of mortality in wild birds (passive surveillance) in Catalonia. The objective is to provide an early warning system to prevent the infection of poultry. Since 2010, 35 episodes of mortality in wild birds were attributed to NDV infection. Throughout this period there was a progressive expansion of NDV to new areas, with an increase in the episodes of mortality, although it is not clear whether they were the result of the spread of the virus, or of the improvement of the surveillance. Phylogenetic analyses indicate that two distinct sublineages of NDV, 4a and 4b, were circulating in Catalonia. Both sublineages seem to be endemic in the wild bird population, affecting mainly Eurasian-collared doves, with a clear pattern in relation to its spatial distribution (coincident with the distribution of this species), and its temporal distribution (with the majority of cases between September and February). So far, endemicity in wild birds has not resulted in ND outbreaks in poultry. However, there are still many uncertainties about, for example, whether NDV may expand to new areas of Catalonia (with higher poultry density), or about the threat that the apparently more novel sublineage 4a may represent. Hence, efforts should be made so that measures to prevent infection of poultry farms (particularly in high-risk areas and periods) are encouraged, and surveillance is maintained.

Modification of graphene oxide by laser irradiation: a new route to enhance antibacterial activity.

The antibacterial activity and possible toxicity of graphene oxide and laser-irradiated graphene oxide (iGO) were investigated. Antibacterial activity was tested on Escherichia coli and shown to be higher for GO irradiated for at least three hours, which seems to be correlated to the resulting morphology of laser-treated GO and independent of the kind and amount of oxygen functionalities. X-ray photoelectron spectroscopy, Raman spectroscopy, dynamic light scattering and scanning electron microscopy (SEM) show a reduction of the GO flakes size after visible laser irradiation, preserving considerable oxygen content and degree of hydrophilicity. SEM images of the bacteria after the exposure to the iGO flakes confirm membrane damage after interaction with the laser-modified morphology of GO. In addition, a fish embryo toxicity test on zebrafish displayed that neither mortality nor sublethal effects were caused by the different iGO solutions, even when the concentration was increased up to four times higher than the one effective in reducing the bacteria survival. The antibacterial properties and the absence of toxicity make the visible laser irradiation of GO a promising option for water purification applications.

Monitoring the Spread of Swine Enteric Coronavirus Diseases in the United States in the Absence of a Regulatory Framework.

The reporting and monitoring of swine enteric coronavirus diseases (SECD), including porcine epidemic diarrhea virus and porcine delta coronavirus, in the United States have been challenging because of the initial absence of a regulatory framework and the emerging nature of these diseases. The National Animal Health Laboratory Network, the Emergency Management and Response System, and the Swine Health Monitoring Project were used to monitor the disease situation between May 2013 and March 2015. Important differences existed between and among them in terms of nature and extent of reporting. Here, we assess the implementation of these systems from different perspectives, including a description and comparison of collected data, disease metrics, usefulness, simplicity, flexibility, acceptability, representativeness, timeliness, and stability. This assessment demonstrates the limitations that the absence of premises identification imposes on certain animal health surveillance and response databases, and the importance of federally regulated frameworks in collecting accurate information in a timely manner. This study also demonstrates the value that the voluntary and producer-organized systems may have in monitoring emerging diseases. The results from all three data sources help to establish the baseline information on SECD epidemiological dynamics after almost 3 years of disease occurrence in the country.

Exploring the surveillance potential of mortality data: nine years of bovine fallen stock data collected in Catalonia (Spain).

The potential of fallen stock data to monitor the health status of animal populations has been noted in previous studies. However, further research is required to implement these systems for surveillance. This work presents a novel approach to determining the baselines associated with bovine fallen stock, comparing patterns between subpopulations and identifying subpopulations in which an abnormal event may occur. This study was based on data from 193,873 disposal visits carried out between 2004 and 2012 across a total of 2,991 bovine farms. Proxy measurements such as the number of collections carried out and the weight of carcasses collected were used. Both outcomes were aggregated weekly at different geographical scales for three production types (beef cattle, dairy cattle and heifer fattening). The analysis of these data combined autoregressive integrated moving average modelling and hierarchical time series methods.The three production types exhibited historical baselines that differed notably from one another. Based on the 757 beef cattle farms monitored, the mean number of collections registered per week at the regional level was 37 (range: 10-83). This series was relatively constant over time and showed a marked yearly seasonality. In contrast, for the 426 dairy cattle farms the mean number of disposal visits registered weekly was 121 (range: 71-180), showing half-yearly and yearly seasonality and a marked increase over the period monitored. From the 1,808 heifer fattening farms the mean number of disposal visits was 248 (range: 166-357) and the pattern presented a marked alternating trend over time. These patterns were assessed and compared at regional, provincial, county and municipal levels. The use of hierarchical time series approaches appeared to be a useful tool for comparing the patterns within different subpopulations over time as well as for assessing the spatial extent to which various abnormal events could be detected.

Evaluation of different enzyme-linked immunosorbent assays for the diagnosis of brucellosis due to Brucella melitensis in sheep.

Six serological assays for the diagnosis of ovine brucellosis, due to Brucella melitensis were evaluated. Reference serum samples from sheep of known B. melitensis infection status (n=118) were assessed using the Rose Bengal test (RBT), complement fixation test (CFT) and four commercial enzyme-linked immunosorbent assays (ELISAs), including two indirect ELISAs (iELISAs), one competitive ELISA (cELISA) and one blocking ELISA (bELISA). The highest differential positive rates (DPR) were obtained with the cELISA and bELISA, while the lowest DPR was estimated using iELISAs. A latent class analysis was performed to estimate the accuracy of the CFT, RBT and bELISA using 1827 sera from sheep undergoing testing as part of a surveillance and control programme. Lower sensitivity and specificity were obtained for the three serological tests when the field samples were used. A higher DPR was achieved by the CFT, compared to bELISA and RBT. The results suggest that ELISAs, and particularly the bELISA, might be suitable for inclusion in the European Union legislation on intra-community trade for diagnosing B. melitensis infection in sheep, as it has a similar test performance compared to the RBT.

Ecological factors driving avian influenza virus dynamics in Spanish wetland ecosystems.

Studies exploring the ecological interactions between avian influenza viruses (AIV), natural hosts and the environment are scarce. Most work has focused on viral survival and transmission under laboratory conditions and through mathematical modelling. However, more integrated studies performed under field conditions are required to validate these results. In this study, we combined information on bird community, environmental factors and viral epidemiology to assess the contribution of biotic and abiotic factors in the occurrence of low pathogenic AIV in Spanish wetlands. For that purpose, seven locations in five different wetlands were studied during two years (2007-2009), including seven sampling visits by location. In each survey, fresh faeces (n = 4578) of wild birds and water samples were collected for viral detection. Also, the vegetation structure, water physical properties of wetlands, climatic conditions and wild bird community composition were determined. An overall AIV prevalence of 1.7%±0.4 was detected in faecal samples with important fluctuations among seasons and locations. Twenty-six AIV were isolated from the 78 RRT-PCR positive samples and eight different haemagglutinines and five neuraminidases were identified, being the combination H3N8 the most frequent. Variation partitioning procedures identified the combination of space and time variables as the most important pure factor - independently to other factors - explaining the variation in AIV prevalence (36.8%), followed by meteorological factor (21.5%) and wild bird community composition/vegetation structure (21.1%). These results contribute to the understanding of AIV ecological drivers in Spanish ecosystems and provide useful guidelines for AIV risk assessment identifying potential hotspots of AIV activity.

Model to track wild birds for avian influenza by means of population dynamics and surveillance information.

Design, sampling and data interpretation constitute an important challenge for wildlife surveillance of avian influenza viruses (AIV). The aim of this study was to construct a model to improve and enhance identification in both different periods and locations of avian species likely at high risk of contact with AIV in a specific wetland. This study presents an individual-based stochastic model for the Ebre Delta as an example of this appliance. Based on the Monte-Carlo method, the model simulates the dynamics of the spread of AIV among wild birds in a natural park following introduction of an infected bird. Data on wild bird species population, apparent AIV prevalence recorded in wild birds during the period of study, and ecological information on factors such as behaviour, contact rates or patterns of movements of waterfowl were incorporated as inputs of the model. From these inputs, the model predicted those species that would introduce most of AIV in different periods and those species and areas that would be at high risk as a consequence of the spread of these AIV incursions. This method can serve as a complementary tool to previous studies to optimize the allocation of the limited AI surveillance resources in a local complex ecosystem. However, this study indicates that in order to predict the evolution of the spread of AIV at the local scale, there is a need for further research on the identification of host factors involved in the interspecies transmission of AIV.

Animal diseases caused by orbiviruses, Algeria.

Antibodies against bluetongue virus were detected in cattle, sheep, goats, and camels in Algeria in 2008. Antibodies against epizootic hemorrhagic disease virus were detected in cattle, but antibodies against African horse sickness virus were not detected in horses and mules. Epizootic hemorrhagic disease in northern Africa poses a major risk for the European Union.

Theoretical and experimental approaches to estimate the usefulness of pooled serum samples for the diagnosis of postweaning multisystemic wasting syndrome.

Classical postweaning multisystemic wasting syndrome (PMWS) diagnosis is based on postmortem findings (histopathology plus viral detection in lymphoid tissues). Because one of the major differences between PMWS-affected and nonaffected pigs is Porcine circovirus-2 (PCV-2) load in serum and tissues, real-time quantitative polymerase chain reaction (qPCR) has been suggested as a potential diagnostic technique for the disease. The objective of the present study was to assess the applicability of qPCR to quantify PCV-2 loads in pooled serum samples as an easy-to-use PMWS diagnostic tool at the herd level. The experimental design included two simulation studies with several serum pool sizes from pigs already screened for PMWS (by histopathology and detection of PCV-2 by qPCR). Several qPCR thresholds were defined and validated with experimental pools created in the laboratory. Quantitative PCR on pooled serum samples did not result in a sufficiently reliable alternate method to the classical PMWS diagnosis method based on individual clinical, histopathological, and PCV-2 detection criteria. However, serum pools seemed to be an alternative at a low economic cost for the quantification of PCV-2 loads in suspicious herds. A targeted (including only clinically diseased animals) sampling approach did not give better estimates compared with a random sampling approach.

Quantitative assessment of the probability of bluetongue virus overwintering by horizontal transmission: application to Germany.

Even though bluetongue virus (BTV) transmission is apparently interrupted during winter, bluetongue outbreaks often reappear in the next season (overwintering). Several mechanisms for BTV overwintering have been proposed, but to date, their relative importance remain unclear. In order to assess the probability of BTV overwintering by persistence in adult vectors, ruminants (through prolonged viraemia) or a combination of both, a quantitative risk assessment model was developed. Furthermore, the model allowed the role played by the residual number of vectors present during winter to be examined, and the effect of a proportion of Culicoides living inside buildings (endophilic behaviour) to be explored. The model was then applied to a real scenario: overwintering in Germany between 2006 and 2007. The results showed that the limited number of vectors active during winter seemed to allow the transmission of BTV during this period, and that while transmission was favoured by the endophilic behaviour of some Culicoides, its effect was limited. Even though transmission was possible, the likelihood of BTV overwintering by the mechanisms studied seemed too low to explain the observed re-emergence of the disease. Therefore, other overwintering mechanisms not considered in the model are likely to have played a significant role in BTV overwintering in Germany between 2006 and 2007.

Persistence of highly pathogenic avian influenza virus (H7N1) in infected chickens: feather as a suitable sample for diagnosis.

Selection of an ideal sample is a vital element in early detection of influenza infection. Rapid identification of infectious individuals or animals is crucial not only for avian influenza virus (AIV) surveillance programmes, but also for treatment and containment strategies. This study used a combination of quantitative real-time RT-PCR with an internal positive control and a cell-titration system to examine the presence of virus in different samples during active experimental AIV infection and its persistence in the infected carcasses. Oropharyngeal/cloacal swabs as well as feather pulp and blood samples were collected from 15-day-old chicks infected with H7N1 highly pathogenic AIV (HPAIV) and the kinetics of virus shedding during active infection were evaluated. Additionally, several samples (muscle, skin, brain, feather pulp and oropharyngeal and cloacal swabs) were examined to assess the persistence of virus in the HPAIV-infected carcasses. Based on the results, feather pulp was found to be the best sample to detect and isolate HPAIV from infected chicks from 24 h after inoculation onwards. Kinetic studies on the persistence of virus in infected carcasses revealed that tissues such as muscle could potentially transmit infectious virus for 3 days post-mortem (p.m.), whilst other tissues such as skin, feather pulp and brain retained their infectivity for as long as 5-6 days p.m. at environmental temperature (22-23 degrees C). These results strongly favour feather as a useful sample for HPAIV diagnosis in infected chickens as well as in carcasses.

Influenza A virus subtypes in wild birds in North-Eastern Spain (Catalonia).

Since the spread of H5N1 highly pathogenic avian influenza virus in 2005, many surveillance programmes have been initiated in poultry and wild birds worldwide. This study describes for the first time the detection of different subtypes of avian influenza viruses (AIV) in wild birds in the West Mediterranean area (Catalonia, North-Eastern Spain). During a 3-year period (from mid-2006 to mid-2009), 1374 birds from 16 different families were examined, and a total of 62 AIV were detected by means of a real-time reverse transcriptase PCR assay. AIV were more frequently detected in Anatidae, Phoenicopteridae, Rallidae and Laridae families. Of the 62 positive samples, 28 AIV could be isolated in embryonated eggs. All isolates were subtyped by haemagglutinin and neuraminidase inhibition techniques and 10 different haemagglutinins (HA) and 7 neuraminidases (NA) were found in 13 different subtype combinations. The most common combinations were H4N6 (22.2%) and H1N1 (18.5%). The HA and NA gene sequences of different AIV subtypes were compared and aligned with those available AIV strains from genome databases. Our studies on AIV phylogenetic analysis revealed that all AIV genes sequenced from wild birds in North-Eastern Spain clustered within Eurasian avian clades, including the sequences of H8, N4 and N5 genes analyzed for the first time in Europe. The results contribute to the understanding of AIV in the Mediterranean area and in Europe.

Evolution of the BSE epidemic in Catalonia (1990-2015) based on a stochastic model.

A stochastic model was used to estimate the number of bovine spongiform encephalopathy (BSE) cases that may have occurred in Catalonia (north-east Spain) from January 1990 to December 2015. The model allowed the evolution of the unobserved cases to be estimated differentiating the population into two subgroups: (1) livestock slaughtered for human consumption without clinical signs and (2) cattle at increased risk (including fallen stock, clinical suspects, emergency slaughtered cattle and cattle with clinical signs at ante mortem inspection). If the first bovine affected by BSE had been introduced into the region in 1986, the model predicted that during the period of study the surveillance system would detect a median of 197 outbreaks (2.5-97.5 percentiles: 157-245). The estimated median number of outbreaks that may have appeared before the establishment of the monitoring program was 80 (2.5-97.5 percentiles: 35-152), and the number of cases would have peaked in 2001.