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Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are ...

North Carolina State University

This study evaluated the seropositivity of Brucella abortus and Leptospira interrogans in ewes with reproductive disturbances in southern Brazil and verified the creatine kinase (CK) activity and oxidation status via assessment of superoxide dismutase, glutathione peroxidase and glutathione transferase in serum of seropositive animals for L. interrogans serovar Icterohaemorrhagiae. For Leptospira infection 381 animals with clinical history of reproductive disturbance from Planalto Serrano de Santa Catarina (Brazil) were analyzed, showing an occurrence for L. interrogans of 20.2% from which 81.8% were seropositive for L. interrogans Icterohaemorrhagiae. Serovars Wolfii, Grippothyphosa, Bratislava, Canicola and Butembo were also identified. In the case of B. abortus, positive cases were identified by buffered acidified antigen, finding 14 positive samples, but none of them were positive after a second test (2-mercaptoethanol), showing the absence of relationship between infection with B. abortus and abortion in the tested individuals. Serum reactive oxygen species (ROS) levels and CK activity were found higher in animals positive for Leptospira infection, presenting higher titrations (1:320) than non-infected individuals. Serum glutathione peroxidase activity was higher in positive animals with titrations 1:160 and 1:320, while serum glutathione S-transferase was higher in positive individuals only for titrations 1:320. Serum superoxide dismutase showed lower activity in infected animals with titrations of 1:320. Our results show the region of Planalto Serrano de Santa Catarina with a high occurrence levels of sheep infected by L. interrogans serovar Icterohaemorrhagiae, from which animals with high titrations (1:320) present oxidative stress elicited by excessive ROS production, triggering the stimulation of antioxidant systems to counter this excess. In summary, ovine with higher titrations (1:320) present oxidative damage that can contribute to disease pathophysiology.

Relation of reproductive disturbance in sheep and Leptospira interrogans serovar Icterohaemorrhagiae infection: Impacts on cellular oxidation status.

Leptospirosis is a bacterial disease that represents a major problem in animal and public health due to its high prevalence and widespread distribution. This zoonotic disease is most prevalent in tropical environments where conditions favour pathogen survival. The ecological preferences of Leptospira serovars are poorly understood, limiting our knowledge of where and when outbreaks can occur, which may result in misinformed prevention and control plans. While the disease can occur consistently in time and space in tropical regions, research on the ecology of leptospirosis remains limited in subtropical regions. This research gap regarding Leptospira ecology brings public and veterinary health problems, impacting local economies. To fill this gap of knowledge, we suggest to assess geographic and ecological features among Leptospira serovars in a subtropical area of Brazil where leptospirosis is endemic to (a) highlight environmental conditions that facilitate or limit Leptospira spread and survival and (b) reconstruct its geographic distribution. An ecological niche modelling framework was used to characterize and compare Leptospira serovars in both geographic and environmental space. Our results show that despite the geographic overlap exhibited by the different serovars assessed, we found ecological divergence among their occupied ecological niches. Ecological divergences were expressed as ranges of potential distributions and environmental conditions found suitably by serovar, Sejroe being the most asymmetric (<0.15). Most important predictors for the potential distribution of most serovars were soil pH (31.7%) and landscape temperature (24.2%). Identification of environmental preferences will allow epidemiologists to better infer the presence of a serovar based on the environmental characteristics of regions rather than inferences based solely on historical epidemiological records. Including geographic and ecological ranges of serovars also may help to forecast transmission potential of Leptospira in public health and the food animal practice.

Spatial distribution and spread potential of sixteen Leptospira serovars in a subtropical region of Brazil.

Nairobi Sheep Disease virus (NSDv) is a zoonotic and tick-borne disease that can cause over 90% mortality in small ruminants. NSDv has historically circulated in East Africa and has recently emerged in the Asian continent. Despite efforts to control the disease, some regions, mostly in warmer climates, persistently report disease outbreaks. Consequently, it is necessary to understand how environmental tolerances and factors that influence transmission may shed light on its possible emergence in other regions. In this study, we quantified the available literature of NSDv from which occurrence data was extracted. In total, 308 locations from Uganda, Kenya, Tanzania, Somalia, India, Sri Lanka and China were coupled with landscape conditions to reconstruct the ecological conditions for NSDv circulation and identify areas of potential disease transmission risk. Our results identified areas suitable for NSDv in Ethiopia, Malawi, Zimbabwe, Southeastern China, Taiwan, and Vietnam. Unsuitable areas included Democratic Republic of Congo, Zambia, and Southern Somalia. In summary, soil moisture, livestock density, and precipitation predispose certain areas to NSDv circulation. It is critical to investigate the epidemiology of NSDv in order to promote better allocation of resources to control its spread in regions that are more at risk. This will help reduce disease impact worldwide as climate change will favor emergence of such vector-borne diseases in areas with dense small ruminant populations.

Nairobi Sheep Disease Virus: A Historical and Epidemiological Perspective.

Influenza viruses (IV) are one of the major threats to human and animal health worldwide due to the variety of species they affect. Pigs play an important role in IV ecology as the "mixing vessel," since they can be infected by swine, avian and human IV, allowing the appearance of new subtypes. Human viruses originated in swine are known as IV of swine origin or swine influenza virus (SwIV) variants. In this study, we identified knowledge tendencies of SwIV and assessed potential bias in the literature caused by these variants. We identified the most mentioned SwIV variants and manually reviewed the literature to determine the number of publications applying the whole influenza nomenclature, a partial nomenclature, only the subtype or mixed terminology, along with the proportion of articles in which the GenBank ID number was available. We observed that the 2009 H1N1 human pandemic created an important bias in SwIV research driven by an increase in human publications on the IV of swine origin. H1N1 is the most studied subtype for swine and humans, followed by H3N2. We found differences between the nomenclatures applied, where partial classifications were slightly more common. Finally, from all the publications, only 25% stated the GenBank ID of the sequence studied. This review represents the most complete exploration of trends in SwIV knowledge to date and will serve as a guidance for future search strategies in SwIV research.

A Century of Swine Influenza: Is It Really Just about the Pigs?

Equine infectious anemia virus (EIAV) is a transboundary disease affecting a large number of equines worldwide. In this study, we assessed the transmission risk of EIAV in Rio Grande do Sul, Brazil. Serum samples from 1010 animals from 341 farms were initially analyzed using agar gel immunodiffusion to detect viral antibodies, and no antibody-positive animals were found. A risk assessment stochastic model was applied to generate the expected number of potential infections per month and to estimate the time to new infections. Our results estimated 6.5 months as the interval for new infections in the worst-case scenario. Among the variables evaluated, the number of transported animals and the test sensitivity influenced the model the most. These results were then used to revisit the impact of EIAV control regulations, which triggered a change in the diagnostic testing required for animal movement, in which the validity of a negative test for EIAV was extended from 60 to 180 days. Finally, revisiting the annual average of infected farms before and after the new regulation, the number of infected farms increased pre-implementation, and then, the number of culled animals increased, which should impact future EIAV incidence in this region. Our results demonstrated the importance of constant reviews of disease control programs and provided quantitative-based knowledge for decision-makers in official veterinary services.

Impact of changes of horse movement regulations on the risks of equine infectious anemia: A risk assessment approach.

The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV - the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains - one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28-35 dpi with sporadic rebounds thereafter. The local IgA response started 4-7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28-35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4-7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels - by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.

The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

Diversity, ecology, and evolution of viruses are commonly determined through phylogenetics, an accurate tool for the identification and study of lineages with different pathological characteristics within the same species. In the case of PRRSV, evolutionary research has divided into two main branches based on the use of a specific gene (i.e., ORF5) or whole genome sequences as the input used to produce the phylogeny. In this study, we performed a review on PRRSV phylogenetic literature and characterized the spatiotemporal trends in research of single gene vs. whole genome evolutionary approaches. Finally, using publicly available data, we produced a Bayesian phylodynamic analysis following each research branch and compared the results to determine the pros and cons of each particular approach. This study provides an exploration of the two main phylogenetic research lines applied for PRRSV evolution, as well as an example of the differences found when both methods are applied to the same database. We expect that our results will serve as a guidance for future PRRSV phylogenetic research.

Whole Genome or Single Genes? A Phylodynamic and Bibliometric Analysis of PRRSV.

Fabrics are widely used in hospitals and many other settings for bedding, clothing, and face masks; however, microbial pathogens can survive on surfaces for a long time, leading to microbial transmission. Coatings of metallic particles on fabrics have been widely used to eradicate pathogens. However, current metal particle coating technologies encounter numerous issues such as nonuniformity, processing complexity, and poor adhesion. To overcome these issues, an easy-to-control and straightforward method is reported to coat a wide range of fabrics by using gallium liquid metal (LM) particles to facilitate the deposition of liquid metal copper alloy (LMCu) particles. Gallium particles coated on the fabric provide nucleation sites for forming LMCu particles at room temperature via galvanic replacement of Cu ions. The LM helps promote strong adhesion of the particles to the fabric. The presence of the LMCu particles can eradicate over 99% of pathogens (including bacteria, fungi, and viruses) within 5 min, which is significantly more effective than control samples coated with only Cu. The coating remains effective over multiple usages and against contaminated droplets and aerosols, such as those encountered in facemasks. This facile coating method is promising for generating robust antibacterial, antifungal, and antiviral fabrics and surfaces.

Alba Frias-De-Diego

College of Veterinary Medicine,
Department of Population Health and Pathobiology,
College of Veterinary Medicine, Department of Population Health and Pathobiology

North Carolina State University

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

Alba Frias-De-Diego

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}College of Veterinary Medicine,Department of Population Health and Pathobiology,College of Veterinary Medicine, Department of Population Health and Pathobiology

North Carolina State University

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays

College of Veterinary Medicine,
Department of Population Health and Pathobiology,
College of Veterinary Medicine, Department of Population Health and Pathobiology