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Pallabi Sil Paul,1 Mallesh Rathnam,1 Aria Khalili,2 Leonardo M Cortez,1 Mahalashmi Srinivasan,3 Emmanuel Planel,4 Jae-Young Cho,2,5 Holger Wille,3 Valerie L Sim,1 Sue-Ann Mok,3 Satyabrata Kar1 1Department of Medicine (Neurology), Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada; 2Quantum and Nanotechnology Research Centre, National Research Council Canada, Edmonton, Alberta, Canada; 3Department of Biochemistry, Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada; 4Department of Psychiatry and Neurosciences, University of Laval, Quebec, Canada; 5Department of Mechanical Engineering, University of Alberta, Edmonton, Alberta, CanadaCorrespondence: Satyabrata Kar, Department of Medicine (Neurology), Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, T6G 2M8, Canada, Tel +1-780-492-9357, Fax +1-780-492-9352, Email skar@ualberta.caIntroduction: Hyperphosphorylation and aggregation of the microtubule-associated tau protein, which plays a critical role in many neurodegenerative diseases (ie, tauopathies) including Alzheimer’s disease (AD), are known to be regulated by a variety of environmental factors including temperature. In this study we evaluated the effects of FDA-approved poly (D,L-lactide-co-glycolic) acid (PLGA) nanoparticles, which can inhibit amyloid-β aggregation/toxicity in cellular/animal models of AD, on temperature-dependent aggregation of 0N4R tau isoforms in vitro.Methods: We have used a variety of biophysical (Thioflavin T kinetics, dynamic light scattering and asymmetric-flow field-flow fractionation), structural (fluorescence imaging and transmission electron microscopy) and biochemical (Filter-trap assay and detection of soluble protein) approaches, to evaluate the effects of native PLGA nanoparticles on the temperature-dependent tau aggregation.Results: Our results show that the aggregation propensity of 0N4R tau increases significantly in a dose-dependent manner with a rise in temperature from 27°C to 40°C, as measured by lag time and aggregation rate. Additionally, the aggregation of 2N4R tau increases in a dose-dependent manner. Native PLGA significantly inhibits tau aggregation at all temperatures in a concentration-dependent manner, possibly by interacting with the aggregation-prone hydrophobic hexapeptide motifs of tau. Additionally, native PLGA is able to trigger disassembly of preformed 0N4R tau aggregates as a function of temperature from 27°C to 40°C.Conclusion: These results, taken together, suggest that native PLGA nanoparticles can not only attenuate temperature-dependent tau aggregation but also promote disassembly of preformed aggregates, which increased with a rise of temperature. Given the evidence that temperature can influence tau pathology, we believe that native PLGA may have a unique potential to regulate tau abnormalities associated with AD-related pathology. Keywords: tau protein, PLGA nanoparticles, tau pathology, protein aggregation

Abstract Background Malaria community case management (CCM) can improve timely access to healthcare, and CCM programmes in sub-Saharan Africa are expanding from serving children under 5 years (CU5) only to all ages. This report characterizes malaria case management in the setting of an age-expanded CCM programme in Chadiza District, Zambia. Methods Thirty-three households in each of 73 eligible communities were randomly selected to participate in a household survey preceding a trial of proactive CCM (NCT04839900). All household members were asked about fever in the prior two weeks and received a malaria rapid diagnostic test (RDT); those reporting fever were asked about healthcare received. Weighted population estimates were calculated and mixed effects regression was used to assess factors associated with malaria care seeking. Results Among 11,030 (98.6%) participants with RDT results (2,357 households), parasite prevalence was 19.1% by RDT; school-aged children (SAC, 5–14 years) had the highest prevalence (28.8%). Prior fever was reported by 12.4% of CU5, 7.5% of SAC, and 7.2% of individuals ≥ 15 years. Among those with prior fever, 34.0% of CU5, 56.0% of SAC, and 22.6% of individuals ≥ 15 years had a positive survey RDT and 73.7% of CU5, 66.5% of SAC, and 56.3% of individuals ≥ 15 years reported seeking treatment; 76.7% across all ages visited a CHW as part of care. Nearly 90% (87.8%) of people who visited a CHW reported a blood test compared with 73.5% seen only at a health facility and/or pharmacy (p

Abstract Background Access to healthcare is important in controlling malaria burden and, as a result, distance or travel time to health facilities is often a significant predictor in modelling malaria prevalence. Adding new health facilities may reduce overall travel time to health facilities and may decrease malaria transmission. To help guide local decision-makers as they scale up community-based accessibility, the influence of the spatial allocation of new health facilities on malaria prevalence is evaluated in Bunkpurugu-Yunyoo district in northern Ghana. A location-allocation analysis is performed to find optimal locations of new health facilities by separately minimizing three district-wide objectives: malaria prevalence, malaria incidence, and average travel time to health facilities. Methods Generalized additive models was used to estimate the relationship between malaria prevalence and travel time to the nearest health facility and other geospatial covariates. The model predictions are then used to calculate the optimisation criteria for the location-allocation analysis. This analysis was performed for two scenarios: adding new health facilities to the existing ones, and a hypothetical scenario in which the community-based healthcare facilities would be allocated anew. An interactive web application was created to facilitate efficient presentation of this analysis and allow users to experiment with their choice of health facility location and optimisation criteria. Results Using malaria prevalence and travel time as optimisation criteria, two locations that would benefit from new health facilities were identified, regardless of scenarios. Due to the non-linear relationship between malaria incidence and prevalence, the optimal locations chosen based on the incidence criterion tended to be inequitable and was different from those based on the other optimisation criteria. Conclusions This study findings underscore the importance of using multiple optimisation criteria in the decision-making process. This analysis and the interactive application can be repurposed for other regions and criteria, bridging the gap between science, models and decisions.

Abstract Background Bayesian methods have been used to generate country-level and global maps of malaria prevalence. With increasing availability of detailed malaria surveillance data, these methodologies can also be used to identify fine-scale heterogeneity of malaria parasitaemia for operational prevention and control of malaria. Methods In this article, a Bayesian geostatistical model was applied to six malaria parasitaemia surveys conducted during rainy and dry seasons between November 2010 and 2013 to characterize the micro-scale spatial heterogeneity of malaria risk in northern Ghana. Results The geostatistical model showed substantial spatial heterogeneity, with malaria parasite prevalence varying between 19 and 90%, and revealing a northeast to southwest gradient of predicted risk. The spatial distribution of prevalence was heavily influenced by two modest urban centres, with a substantially lower prevalence in urban centres compared to rural areas. Although strong seasonal variations were observed, spatial malaria prevalence patterns did not change substantially from year to year. Furthermore, independent surveillance data suggested that the model had a relatively good predictive performance when extrapolated to a neighbouring district. Conclusions This high variability in malaria prevalence is striking, given that this small area (approximately 30 km × 40 km) was purportedly homogeneous based on country-level spatial analysis, suggesting that fine-scale parasitaemia data might be critical to guide district-level programmatic efforts to prevent and control malaria. Extrapolations results suggest that fine-scale parasitaemia data can be useful for spatial predictions in neighbouring unsampled districts and does not have to be collected every year to aid district-level operations, helping to alleviate concerns regarding the cost of fine-scale data collection.

Abstract Background Since 2008 indoor residual spraying (IRS) has become one of the interventions for malaria control in Ghana. Key partners in the scale-up of IRS have been the US President’s Malaria Initiative (PMI) and AngloGold Ashanti (AGA). This study was designed to assess the impact of IRS on malaria parasitaemia among children less than 5 years-old in Bunkpurugu-Yunyoo, one of PMI-sponsored districts in northern Ghana, where rates of parasitaemia significantly exceeded the national average. Methods Two pre-IRS cross-sectional surveys using microscopy were conducted in November 2010 and April 2011 to provide baseline estimates of malaria parasitaemia for the high and low transmission seasons, respectively. IRS for the entire district was conducted in May/June to coincide with the beginning of the rains. Alpha-cypermethrin was used in 2011 and 2012, and changed to pirimiphos-methyl in 2013 and 2014 following declining susceptibility of local vectors to pyrethroids. Post-IRS cross-sectional surveys were conducted between 2011 and 2014 to provide estimates for the end of high (2011–2014) and the end of low (2012–2013) transmission seasons. Results The end of high transmission season prevalence of asexual parasitaemia declined marginally from 52.4% (95% CI: 50.0–54.7%) to 47.7% (95% CI: 45.5–49.9%) following 2 years of IRS with alpha-cypermethrin. Prevalence declined substantially to 20.6% (95% CI: 18.4–22.9%) following one year of IRS with pirimiphos-methyl. Conclusions The use of a more efficacious insecticide for IRS can reduce malaria parasitaemia among children less than 5 years-old in northern Ghana.

Abstract Background There is a need for comprehensive evaluations of the underlying local factors that contribute to residual malaria in sub-Saharan Africa. However, it is difficult to compare the wide array of demographic, socio-economic, and environmental variables associated with malaria transmission using standard statistical approaches while accounting for seasonal differences and nonlinear relationships. This article uses a Bayesian model averaging (BMA) approach for identifying and comparing potential risk and protective factors associated with residual malaria. Results The relative influence of a comprehensive set of demographic, socio-economic, environmental, and malaria intervention variables on malaria prevalence were modelled using BMA for variable selection. Data were collected in Bunkpurugu-Yunyoo, a rural district in northeast Ghana that experiences holoendemic seasonal malaria transmission, over six biannual surveys from 2010 to 2013. A total of 10,022 children between the ages 6 to 59 months were used in the analysis. Multiple models were developed to identify important risk and protective factors, accounting for seasonal patterns and nonlinear relationships. These models revealed pronounced nonlinear associations between malaria risk and distance from the nearest urban centre and health facility. Furthermore, the association between malaria risk and age and some ethnic groups was significantly different in the rainy and dry seasons. BMA outperformed other commonly used regression approaches in out-of-sample predictive ability using a season-to-season validation approach. Conclusions This modelling framework offers an alternative approach to disease risk factor analysis that generates interpretable models, can reveal complex, nonlinear relationships, incorporates uncertainty in model selection, and produces accurate predictions. Certain modelling applications, such as designing targeted local interventions, require more sophisticated statistical methods which are capable of handling a wide range of relevant data while maintaining interpretability and predictive performance, and directly characterize uncertainty. To this end, BMA represents a valuable tool for constructing more informative models for understanding risk factors for malaria, as well as other vector-borne and environmentally mediated diseases.

This paper presents results of singlet oxygen generation experiments in a high-pressure, non-self-sustained crossed discharge. The discharge consists of a high-voltage, short pulse duration, high repetition rate pulsed discharge, which produces ionization in the flow, and a low-voltage dc discharge which sustains current in a decaying plasma between the pulses. The sustainer voltage can be independently varied to maximize the energy input into electron impact excitation of singlet delta oxygen (SDO). The results demonstrate operation of a stable and diffuse crossed discharge in O2–He mixtures at static pressures of at least up to P0 = 380 Torr and sustainer discharge powers of at least up to 1200 W, achieved at P0 = 120 Torr. The reduced electric field in the positive column of the sustainer discharge varies from E/N = 0.3 10−16 to 0.65 10−16 V cm2, which is significantly lower than E/N in self-sustained discharges and close to the theoretically predicted optimum value for O2(a 1Δ) excitation. Measurements of visible emission spectra O2(b 1Σ → X 3Σ) in the discharge afterglow show the O2(b 1Σ) concentration to increase with the sustainer discharge power and to decrease as the O2 fraction in the flow is increased. Rotational temperatures inferred from these spectra in 10% O2–90% He flows at P0 = 120 Torr and mass flow rates of are 365–465 K. SDO yield at these conditions, 1.7% to 4.4%, was inferred from the integrated intensity of the (0, 0) band of the O2(a 1Δ → X 3Σ) infrared emission spectra calibrated using a blackbody source. The yield remains nearly constant in the discharge afterglow, up to at least 15 cm distance from the discharge. Kinetic modelling calculations using a quasi-one-dimensional nonequilibrium pulser–sustainer discharge model coupled with the Boltzmann equation for plasma electrons predict gas temperature rise in the discharge in satisfactory agreement with the experimental measurements. However, the model overpredicts the O2(a 1Δ) yield by a factor of 2–2.5, which suggests that the model''s description of nonequilibrium O2–He plasma kinetics at high pressures is not quite adequate. [ABSTRACT FROM AUTHOR]

Abstract Background Indoor residual spraying (IRS) is being implemented as one of the malaria prevention methods in the Northern Region of Ghana. Changes in longevity, sporozoite and entomological inoculation rates (EIRs) of major malaria vectors were monitored to assess the impact of IRS in selected districts. Methods Monthly human landing catches (HLCs) were used to collect mosquitoes from sentinel sites in three adjacent districts between July 2009 and December 2014: Savelugu Nanton (SND) where IRS had been implemented from 2008 to 2014; Tolon Kumbungu (TKD) where IRS had been implemented between 2008 and 2012 and Tamale Metropolis (TML) with no history of IRS. Mosquitoes were morphologically identified to species level and into sibling species, using PCR. Samples of Anopheles gambiae sensu lato (s.l.) were examined for parity and infectivity. EIR was calculated from biting and infectivity rates of malaria vectors. Results Parity rates of An. gambiae s.l. decreased significantly (p

This study aims to evaluate the aerobic fitness of miners and develop a predictive equation to assess their maximum oxygen uptake ( V O2max ), which is crucial for ensuring their safety and health in harsh working conditions. Measurement of V O2max via indirect calorimetry is costly and requires skilled personnel; developing a predictive equation is a viable alternative. Existing predictive equations, e.g., Hunt, Kline and Dolgener equations, were tested using the Pearson correlation coefficient, constant error, standard error of estimate and total error. The aerobic fitness of shearer operators, bolters miners, jackhammer drillers and road header operators was very low. Bland-Altman analysis revealed that predicted V O2max measurements of all three equations had a total error of >10%, which is unacceptable for usage. Therefore, a new predictive equation was developed with a total error of 9.78%. The developed predictive equation is useful to evaluate miners' functional capacity with higher precision and assist in allocating work.

One of the most vital parameters to achieve sustainability in any field is encompassing the Occupational Health and Safety (OHS) of the workers. In mining industry where heavy earth moving machineries are largely employed, ergonomic hazards turn out to be significant OHS hazards causing Musculoskeletal Disorders (MSDs) in the operators. Nevertheless, the Indian mining industry lacks a comprehensive technique of OHS risk assessment, especially for ergonomic hazards that cause MSDs. This research appraises ergonomic hazards and develops Fuzzy Musculoskeletal-disorders Index (FMI) model to evaluate ergonomic-related MSDs. Work process and work tool ergonomic risk factors were identified through literature review and directives recommended by experts. Work posture was evaluated using RULA. The data-collecting approach was implemented using participatory ergonomic and design science principles. The FMI results show average MSDs score of 3.69, indicating high to extremely high risk. Surface plots show that combined work tool and work process was the most sensitive factors to MSDs risk compared to other two combinations. A two-sample t-test validated the FMI. The findings should help safety experts and managers develop effective OHS management plans and programmes for the sustainability of Indian mining industry., (© 2024. The Author(s).)

Evidence suggests that beta-amyloid (Aβ)-induced phosphorylation/aggregation of tau protein plays a critical role in the degeneration of neurons and development of Alzheimer's disease (AD), the most common cause of dementia affecting the elderly population. Many studies have pursued a variety of small molecules, including nanoparticles conjugated with drugs to interfere with Aβ and/or tau aggregation/toxicity as an effective strategy for AD treatment. We reported earlier that FDA approved PLGA nanoparticles without any drug can attenuate Aβ aggregation/toxicity in cellular/animal models of AD. In this study, we evaluated the effects of native PLGA on Aβ seed-induced aggregation of tau protein using a variety of biophysical, structural and spectroscopic approaches. Our results show that Aβ 1-42 seeds enhanced aggregation of tau protein in the presence and absence of heparin and the effect was attenuated by native PLGA nanoparticles. Interestingly, PLGA inhibited aggregation of both 4R and 3R tau isoforms involved in the formation of neurofibrillary tangles in AD brains. Furthermore, Aβ seed-induced tau aggregation in the presence of arachidonic acid was suppressed by native PLGA. Collectively, our results suggest that native PLGA nanoparticles can inhibit the Aβ seed-induced aggregation of different tau protein isoforms highlighting their therapeutic implication in the treatment of AD., (© 2024. The Author(s).)

Introduction: Spatial analysis of lipids in inflammatory microenvironments is key to understand the pathogenesis of infectious disease. Granulomatous inflammation is a hallmark of leishmaniasis and changes in host and parasite lipid metabolism have been observed at the bulk tissue level in various infection models. Here, mass spectrometry imaging (MSI) is applied to spatially map hepatic lipid composition following infection with Leishmania donovani , an experimental mouse model of visceral leishmaniasis., Methods: Livers from naïve and L. donovani -infected C57BL/6 mice were harvested at 14- and 20-days post-infection (n=5 per time point). 12 µm transverse sections were cut and covered with norhamane, prior to lipid analysis using MALDI-MSI. MALDI-MSI was performed in negative mode on a Rapiflex (Bruker Daltonics) at 5 and 50 µm spatial resolution and data-dependent analysis (DDA) on an Orbitrap-Elite (Thermo-Scientific) at 50 µm spatial resolution for structural identification analysis of lipids., Results: Aberrant lipid abundances were observed in a heterogeneous distribution across infected mouse livers compared to naïve mouse liver. Distinctive localized correlated lipid masses were found in granulomas and surrounding parenchymal tissue. Structural identification revealed 40 different lipids common to naïve and d14/d20 infected mouse livers, whereas 15 identified lipids were only detected in infected mouse livers. For pathology-guided MSI imaging, we deduced lipids from manually annotated granulomatous and parenchyma regions of interests (ROIs), identifying 34 lipids that showed significantly different intensities between parenchyma and granulomas across all infected livers., Discussion: Our results identify specific lipids that spatially correlate to the major histopathological feature of Leishmania donovani infection in the liver, viz. hepatic granulomas. In addition, we identified a three-fold increase in the number of unique phosphatidylglycerols (PGs) in infected liver tissue and provide direct evidence that arachidonic acid-containing phospholipids are localized with hepatic granulomas. These phospholipids may serve as important precursors for downstream oxylipin generation with consequences for the regulation of the inflammatory cascade. This study provides the first description of the use of MSI to define spatial-temporal lipid changes at local sites of infection induced by Leishmania donovani in mice., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tans, Dey, Dey, Cao, Paul, Calder, O’Toole, Kaye and Heeren.)

Conversion of β-amyloid (Aβ) peptides from soluble random-coil to aggregated protein enriched with β-sheet-rich intermediates has been suggested to play a role in the degeneration of neurons and development of Alzheimer's disease (AD) pathology. Aggregation of Aβ peptide can be prompted by a variety of environmental factors including temperature which can influence disease pathogenesis. Recently, we reported that FDA-approved unconjugated poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles can have beneficial effects in cellular and animal models of AD by targeting different facets of the Aβ axis. In this study, using biochemical, structural and spectroscopic analyses, we evaluated the effects of native PLGA on temperature-dependent Aβ aggregation and its ability to protect cultured neurons from degeneration. Our results show that the rate of spontaneous Aβ 1-42 aggregation increases with a rise in temperature from 27 to 40 °C and PLGA with 50:50 resomer potently inhibits Aβ aggregation at all temperatures, but the effect is more profound at 27 °C than at 40 °C. It appears that native PLGA, by interacting with the hydrophobic domain of Aβ 1-42 , prevents a conformational shift towards β-sheet structure, thus precluding the formation of Aβ aggregates. Additionally, PLGA triggers disassembly of matured Aβ 1-42 fibers at a faster rate at 40 °C than at 27 °C. PLGA-treated Aβ samples can significantly enhance viability of cortical cultured neurons compared to neurons treated with Aβ alone by attenuating phosphorylation of tau protein. Injection of native PLGA is found to influence the breakdown/clearance of Aβ peptide in the brain. Collectively, these results suggest that PLGA nanoparticles can inhibit Aβ aggregation and trigger disassembly of Aβ aggregates at temperatures outside the physiological range and can protect neurons against Aβ-mediated toxicity thus validating its unique therapeutic potential in the treatment of AD pathology., (© 2022. The Author(s).)

Occupational injuries in mines are attributed to many factors. In this study, an attempt was made to identify the various factors related to work injuries in mines and to estimate their effects on work injuries to mine workers. An accident path model was developed to estimate the pattern and strength of relationships amongst the personal and sociotechnical variables in accident/injury occurrences. The input data for the model were the correlation matrix of 18 variables, which were collected from the case study mines. The case study results showed that there are sequential interactions amongst the sociotechnical and personal factors leading to accidents/injuries in mines. Amongst the latent endogenous constructs, job dissatisfaction and safe work behaviour show a significant positive and negative direct relationship with work injury, respectively. However, the construct safety environment has a significant negative indirect relationship with work injury. The safety environment is negatively affected by work hazards and positively affected by social support. The safety environment also shows a significant negative relationship with job stress and job dissatisfaction. However, negative personality has no significant direct or indirect effect on work injury, but it has a significant negative relationship with safe work behaviour. The endogenous construct negative personality is positively influenced by job stress and negatively influenced by social support.

We assessed 18 children with unilateral amblyopia and 30 age-matched controls on one low-level and three high-level motion tasks. Children with amblyopia showed similar performance to controls in both amblyopic and fellow eyes on a low-level global motion task and on a high-level 2-dot apparent motion task. Performance on both single-object and multiple-object attentive tracking tasks was significantly depressed in both amblyopic and fellow eyes relative to controls. These findings suggest that binocular regions of posterior parietal cortex likely contribute to a deficit in voluntary, spatial attention that is a component of amblyopia.

Type 2 porcine circovirus (PCV2) is associated with postweaning multisystemic wasting syndrome in pigs, whereas the genetically related type 1 PCV (PCV1) is nonpathogenic. In this study, seven monoclonal antibodies (MAbs) against PCV2-ORF2 capsid protein were generated, biologically characterized, and subsequently used to map the antigenic sites of PCV2 capsid protein by using infectious PCV DNA clones containing PCV1/PCV2-ORF2 chimeras. The PCV1/PCV2-ORF2 chimeras were constructed by serial deletions of PCV2-ORF2 and replacement with the corresponding sequences of the PCV1-ORF2. The reactivities of chimeric PCV1/PCV2 clones in transfected PK-15 cells with the seven MAbs were detected by an immunofluorescence assay (IFA). The chimera (r140) with a deletion of 47 amino acids at the N terminus of PCV2-ORF2 reacted strongly to all seven MAbs. Expanding the deletion of PCV2-ORF2 from residues 47 to 57 (r175) abolished the recognition of MAb 3B7, 3C11, 4A10, 6H2, or 8F6 to the chimera. Further deletion of PCV2-ORF2 to 62 residues disrupted the binding of this chimera to all seven MAbs. IFA reactivities with all MAbs were absent when residues 165 to 233 at the C terminus of PCV2-ORF2 was replaced with that of PCV1-ORF2. Extending the sequence of PCV2-ORF2 from residues 165 (r464) to 185 (r526), 200 (r588), or 224 (r652) restored the ability of the three chimeras to react with MAbs 3C11, 6H2, 9H7, and 12G3 but not with 8F6, 3B7, or 4A10. When the four amino acids at the C terminus of r588 were replaced with that of PCV2-ORF2, the resulting chimera (r588F) reacted with all seven MAbs. The results from this study suggest that these seven MAbs recognized at least five different but overlapping conformational epitopes within residues 47 to 63 and 165 to 200 and the last four amino acids at the C terminus of the PCV2 capsid protein.

The pathogenicity of three isolates of porcine respiratory coronavirus (AR310, LEPP and 1894) from the USA was assessed in specific pathogen-free pigs. Pigs inoculated with 1894 developed mild respiratory disease and pigs inoculated with AR310 and LEPP developed moderate respiratory disease from four to 10 days after they were inoculated, but all the pigs recovered fully by 14 days after inoculation. Gross and microscopic examination revealed mild (1894) to moderate (AR310 and LEPP) multifocal bronchointerstitial pneumonia from four to 10 days after inoculation. The lesions were characterised by necrotising bronchiolitis, septal infiltration with mononuclear cells, and a mixed alveolar exudate. No clinical signs or microscopic lesions were observed in control pigs that had not been inoculated.

Porcine organs, cells and tissues provide a viable source of transplants in humans, though there is some concern of public health risk from adaptation of swine infectious agents in humans. Limited information is available on the public health risk of many exogenous swine viruses, and reliable and rapid diagnostic tests are available for only a few of these. The ability of several porcine viruses to cause transplacental fetal infection (parvoviruses, circoviruses, and arteriviruses), emergence or recognition of several new porcine viruses during the last two decades (porcine circovirus, arterivirus, paramyxoviruses, herpesviruses, and porcine respiratory coronavirus) and the immunosuppressed state of the transplant recipients increases the xenozoonoses risk of humans to porcine viruses through transplantation. Much of this risk can be eliminated with vigilance and sustained monitoring along with a better understanding of pathogenesis and development of better diagnostic tests. In this review we present information on selected exogenous viruses, highlighting their characteristics, pathogenesis of viral infections in swine, methods for their detection, and the potential xenozoonoses risk they present. Emphasis has been given in this review to swine influenza virus, paramyxovirus (Nipah virus, Menagle virus, LaPiedad paramyxovirus, porcine paramyxovirus), arterivirus (porcine reproductive and respiratory syndrome virus) and circovirus as either they represent new swine viruses or present the greatest risk. We have also presented information on porcine parvovirus, Japanese encephalitis virus, encephalomyocarditis virus, herpesviruses (pseudorabies virus, porcine lymphotropic herpesvirus, porcine cytomegalovirus), coronaviruses (TGEV, PRCV, HEV, PEDV) and adenovirus. The potential of swine viruses to infect humans needs to be assessed in vitro and in vivo and rapid and more reliable diagnostic methods need to be developed to assure safe supply of porcine tissues and cells for xenotransplantation.

Porcine rotaviruses are a common cause of gastroenteritis. Several serotypes have been detected based on the two surface proteins VP4 (P-types) and VP7 (G-types). However, limited studies have been performed on the relative frequency of rotavirus types in diarrhetic pigs primarily because of the lack of availability of suitable methods. In this study, we describe a reverse transcriptase-polymerase chain reaction (RT-PCR) method for the typing of P and G types of rotavirus. This method allowed to detect G and P types in 96.8 and 87.1% of isolates collected in the United States, respectively and in 54.5 and 38.6% of isolates collected in Poland, respectively. Within the US specimens the G3, G4, G5, G9 and G10 types were detected in combination with P6 and P7 types while among Polish specimens only G3, G4 and G5 types in combination with P6 and P7 types were identified. In both instances the G4 and G5 were the most prevalent types. These studies show that a RT-PCR typing method is suitable for molecular epidemiological studies and that there is more diversity among porcine rotavirus than previously reported.

Seventy five-week-old, crossbred, caesarean-derived, colostrum-deprived pigs were randomly divided into five groups of 14 pigs and assigned one of five treatments: the intranasal inoculation of 1 (5.7) TCID50 of one of four plaque-purified isolates of porcine reproductive and respiratory syndrome virus (PRRSV) (VR2385, VR2431, ISU-984 and ISU-22), or uninfected cell culture and media. Haematological variables were measured for 21 days and bone marrow was analysed when the pigs were killed three, seven, 10, 21 or 28 days after the inoculation. The PRRSV-infected pigs had non-regenerative anaemia and markedly increased myeloid:erythroid ratios from three to 21 days after inoculation. There was a significant (P < 0.05) difference in the severity of the anaemia induced by the four PRRSV isolates; the most highly pneumovirulent strains (VR2385, ISU-984 and ISU-22) induced more severe anaemia than the least virulent isolate (VR2431). The anaemia induced by PRRSV was probably due to a direct or indirect effect on erythroid precursor cells in the bone marrow.

Postweaning multisystemic wasting syndrome of swine associated with porcine circovirus (PCV) is a recently reported and economically important disease. Simple and reliable diagnostic methods are needed for detecting antibodies to PCV type 2 (PCV2) for monitoring of PCV infection. Here, we report the development of two modified indirect enzyme-linked immunosorbent assays (ELISAs): a PCV2 ELISA based on cell-culture-propagated PCV2 and an ORF2 ELISA based on recombinant major capsid protein. PCV2 and ORF2 ELISA detected antibodies to PCV2 and the capsid protein, respectively, in sera from pigs experimentally infected with PCV2 as early as 14 and 21 days postinoculation (dpi). The kinetics of the antibody response to PCV2 and the major capsid protein were similar. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs for both assays were less than 30%. To validate the assays, PCV2 and ORF2 ELISAs were performed with 783 serum samples of young and adult pigs collected from different herds in the Midwestern United States and compared with an indirect immunofluorescent assay (IIF). Six out of 60 samples collected from nursery and growing pigs in 1987 were positive by both ELISA and IIF. Compared with IIF, the diagnostic sensitivity, specificity, and accuracy of PCV2 and ORF2 ELISAs were similar (>90%). The tests showed no cross-reactivity with antibodies to porcine parvovirus and porcine reproductive and respiratory syndrome virus. There was good agreement between the two ELISAs and between the ELISAs and IIF. The availability of the two ELISAs should accelerate our understanding of the host immune response to PCV2 and facilitate the development of prevention and control strategies by elucidating the ecology of PCV2 within swine populations.

In the last few years, newly recognized paramyxoviruses have been associated with severe disease in several animal species, including swine, as well as in human beings. Recently, a paramyxovirus was isolated from a swine herd in the northcentral United States that experienced an epizootic of respiratory and central nervous system disease. Affected pigs had interstitial pneumonia with necrotizing bronchiolitis and encephalitis characterized by lymphocytic perivasculitis and diffuse gliosis. Germ-free pigs inoculated with this isolate developed mild clinical illness and similar but less severe histopathologic lesions in lungs and brain.

Three-week-old cesarean-derived colostrum-deprived (CD/CD) pigs were inoculated with porcine circovirus type 2 (PCV2, n = 19), porcine reproductive and respiratory syndrome virus (PRRSV, n = 13), concurrent PCV2 and PRRSV (PCV2/PRRSV, n = 17), or a sham inoculum (n = 12) to compare the independent and combined effects of these agents. Necropsies were performed at 7, 10, 14, 21, 35, and 49 days postinoculation (dpi) or when pigs became moribund. By 10 dpi, PCV2/PRRSV-inoculated pigs had severe dyspnea, lethargy, and occasional icterus; after 10 dpi, mortality in this group was 10/11 (91%), and all PCV2/ PRRSV-inoculated pigs were dead by 20 dpi. PCV2-inoculated pigs developed lethargy and sporadic icterus, and 8/19 (42%) developed exudative epidermitis; mortality was 5/19 (26%). PRRSV-inoculated pigs developed dyspnea and mild lethargy that resolved by 28 dpi. Microscopic lesions consistent with postweaning multisystemic wasting syndrome (PMWS) were present in both PCV2- and PCV2/PRRSV-inoculated pigs and included lymphoid depletion, necrotizing hepatitis, mild necrotizing bronchiolitis, and infiltrates of macrophages that occasionally contained basophilic intracytoplasmic inclusion bodies in lymphoid and other tissues. PCV2/ PRRSV-inoculated pigs also had severe proliferative interstitial pneumonia and more consistent hepatic lesions. The most severe lesions contained the greatest number of PCV2 antigen-containing cells. PRRSV-inoculated pigs had moderate proliferative interstitial pneumonia but did not develop bronchiolar or hepatic lesions or lymphoid depletion. All groups remained seronegative to porcine parvovirus. The results indicate that 1) PCV2 coinfection increases the severity of PRRSV-induced interstitial pneumonia in CD/CD pigs and 2) PCV2 but not PRRSV induces the lymphoid depletion, granulomatous inflammation, and necrotizing hepatitis characteristic of PMWS.

Commercially available serum replacements are often used in cell culture as a cheaper and less variable substitute for fetal bovine serum (FBS). The growth of porcine reproductive and respiratory syndrome virus (PRRSV) isolates in CRL11171 cells maintained in a medium supplemented with FBS was compared with virus propagation in the same cell line maintained in the same medium with a serum replacement. The PRRSV replicated significantly better when the cell culture medium was supplemented with FBS. The results of this study have implications for the use of serum replacement-supplemented medium for PRRSV diagnosis by virus isolation.

A double in situ hybridization method for the simultaneous detection of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCV) genomes in the same tissue section was applied to lung tissues from 9 pigs in which PRRSV and PCV coinfection had been previously demonstrated. Paraffin-embedded tissue sections were simultaneously hybridized with a digoxigenin-labeled antisense RNA probe for PRRSV and a fluorescein-labeled antisense RNA probe for PCV, and hybridization was detected with anti-digoxigenin alkaline phosphatase/fast red and anti-fluorescein peroxidase/diaminobenzidine, respectively. PRRSV and PCV genomes were identified in the same pulmonary cell types as reported previously in all 9 pigs. In all pigs, PCV-positive cells outnumbered PRRSV-positive cells. A small proportion of alveolar macrophages contained both PRRSV and PCV genomes.

A rapid in situ hybridization (ISH) technique for the detection of porcine circovirus (PCV) nucleic acid in cell culture and formalin-fixed paraffin-embedded tissues was developed. A fluorescein-labeled RNA probe was transcribed from a plasmid containing 530 bp of the ORF1 of a PCV isolated from a pig with postweaning multisystemic wasting syndrome (PMWS). Hybridization using standard hybridization buffer was performed at 42 C for 16 hours and was compared to hybridization using rate enhancement hybridization (REH) buffer at 67 C for 2 hours. Hybridization was detected with an alkaline phosphatase-conjugated antifluorescein antibody. In both cultured cells and tissues from pigs with PMWS, the signal intensity and number of labeled cells in sections hybridized with REH buffer were equal to those of sections hybridized with standard hybridization buffer. The total time required for ISH using the REH buffer is 7-8 hours, thus making this protocol suitable for application in routine PCV diagnosis.

Bluetongue (BT) is an arthropod-borne viral disease affecting ruminants primarily in tropical and temperate regions of the world. Of the 24 serotypes of BT virus (BTV) identified worldwide, five have been found in the United States. Serotype identification of BTV isolates is important to the epidemiology of the virus, but current methods are cumbersome. A single-tube multiplex reverse transcriptase polymerase chain reaction (mRT-PCR) assay, previously developed for the serotype determination of U.S. BTV isolates, was evaluated. The determination of serotype was based on the size of the resultant amplified product. The procedure was evaluated using all 24 serotypes of BTV and nine serotypes of epizootic hemorrhagic disease virus (EHDV), a closely related orbivirus. Only the five U.S. serotypes of BTV were detected by the mRT-PCR. The assay was further tested using 132 BTV isolates originating from 24 western and southern states of the United States, from several different host species, spanning a period of 24 years. The serotypes of the isolates were determined by both a virus neutralization (VN) procedure and the mRT-PCR. Comparison of the mRT-PCR to the standard VN showed that the mRT-PCR successfully identified the serotypes of 130 of the isolates and was shown to be more reliable and specific than the VN assay.

Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.

Porcine circovirus type 1 (PCV1), a PK-15 cell line contaminant, and porcine circovirus type 2 (PCV2), associated with post-weaning multisystemic wasting syndrome (PMWS), are genetically and antigenically related. Several techniques have been developed to detect PCV, including in situ hybridization (ISH). Previously reported probes used for ISH may hybridize with both PCV1 and PCV2 nucleic acids. We attempted to produce probes for ISH that can detect and differentiate PCV2 from PCV1 in PCV-infected cells. Riboprobes were synthesized from the sense and antisense strands of both open reading frames 1 and 2 (ORF1 and ORF2) of PCV2. At 42 and 58 degrees C, the ORF1 antisense probe hybridized with nucleic acid from both PCV1- and PCV2-infected cells. At 58 degrees C, the ORF2 antisense probe hybridized with PCV2 nucleic acid but not with PCV1 nucleic acid. The ORF1 and ORF2 sense probes bound only with PCV2 nucleic acid. Both antisense strand probes produced stronger signals than the sense strand probes. The results showed that the PCV2 ORF1 antisense probe is the most likely probe to detect both PCV types while the ORF2 antisense probe is capable of discriminating between PCV1 and PCV2.

An immunohistochemical method for the detection of type 2 porcine circovirus (PCV2) in paraffin-embedded tissue was developed. Rabbits were inoculated with purified PCV2 to obtain a polyclonal antiserum. Antiserum was applied to sections of porcine tissue that contained lesions consistent with postweaning multisystemic wasting syndrome and in which PCV2 genome had been demonstrated by in situ hybridization. In all cases (18/18), the density and distribution of positive cells detected by in situ hybridization or immunohistochemistry were identical. The immunohistochemical method is more rapid and less expensive than in situ hybridization and is thus more suitable for routine diagnostic use.

Evidence of apoptosis was detected for the United States porcine reproductive and respiratory syndrome virus (PRRSV) in ATCC CRL11171 cells inoculated with strain ATCC VR2385 and in the tissues of pigs infected with the same strain. Apoptosis was detected by agarose gel electrophoresis, transmission electron microscopy and terminal deoxytransferase dUTP nick end labelling (TUNEL) techniques. By electron microscopy and double-labelling techniques, apoptosis was detected primarily in uninfected bystander cells in the continuous cell line rather than the PRRSV-infected cells. In the lungs, the apoptotic cells were predominantly alveolar and pulmonary intravascular macrophages, and mononuclear cells in the alveolar septa. In the lymph nodes, the apoptotic cells were predominantly tingible body macrophages and mononuclear cells. The induction of apoptosis in a large number of mononuclear cells in the lungs and lymph nodes appears to be a mechanism of PRRSV pathogenesis and might be an explanation for a dramatic reduction in the number of alveolar macrophages and circulating lymphocytes and monocytes in PRRSV-infected pigs.

Monoclonal antibodies (MAbs) against porcine reproductive and respiratory syndrome virus (PRRSV) were prepared and characterized. Four MAbs were developed from the mice immunized with the recombinant GP4 protein expressed in insect cells, and six MAbs were derived from the immunization with recombinant GP5 protein. All of the MAbs showed strong perinuclear fluorescence in PRRSV VR2385 infected cells by immunofluorescence staining. Among the MAbs to GP5 protein, one showed strong reactivity in ELISA and recognized a 26 kDa band of PRRSV in a western blot assay, while another showed neutralizing activity against the VR2385 isolate. Out of the four MAbs to GP4 protein, one showed mild reactivity in ELISA with detergent extracted antigen, but had no reactivity in a western-blot assay. The failure of MAb binding to detergent extracted antigen in ELISA or in western-blot analysis indicated that the MAbs were against conformationally dependent epitopes. Reactivity patterns of the MAbs with PRRSV field isolates tested by fixed-cell ELISA showed that there are antigenic variations in PRRSV GP4 and GP5 proteins. Development of these MAbs will benefit further studies on PRRSV structural proteins as well as in understanding their roles in PRRSV pathogenesis.