Development of a novel real-time PCR multiplex assay for detection of Streptococcus equi subspecies equi and Streptococcus equi subspecies zooepidemicus.

Strangles is a contagious bacterial disease of horses caused by Streptococcus equi subspecies equi (SEE) that occurs globally. Rapid and accurate identification of infected horses is essential for controlling strangles. Because of limitations of existing PCR assays for SEE, we sought to identify novel primers and probes that enable simultaneous detection and differentiation of infection with SEE and S. equi subsp. zooepidemicus (SEZ). Comparative genomics of U.S. strains of SEE and SEZ (n = 50 each) identified SE00768 from SEE and comB from SEZ as target genes. Primers and probes for real-time PCR (rtPCR) were designed for these genes and then aligned in silico with the genomes of strains of SEE (n = 725) and SEZ (n = 343). Additionally, the sensitivity and specificity relative to microbiologic culture were compared between 85 samples submitted to an accredited veterinary medical diagnostic laboratory. The respective primer and probe sets aligned with 99.7 % (723/725) isolates of SEE and 97.1 % (333/343) of SEZ. Of 85 diagnostic samples, 20 of 21 (95.2 %) SEE and 22 of 23 SEZ (95.6 %) culture-positive samples were positive by rtPCR for SEE and SEZ, respectively. Both SEE (n = 2) and SEZ (n = 3) were identified by rtPCR among 32 culture-negative samples. Results were rtPCR-positive for both SEE and SEZ in 21 of 44 (47.7 %) samples that were culture-positive for SEE or SEZ. The primers and probe sets reported here reliably detect SEE and SEZ from Europe and the U.S., and permit detection of concurrent infection with both subspecies.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Osteopontin activity modulates sex-specific calcification in engineered valve tissue mimics.

Patients with aortic valve stenosis (AVS) have sexually dimorphic phenotypes in their valve tissue, where male valvular tissue adopts a calcified phenotype and female tissue becomes more fibrotic. The molecular mechanisms that regulate sex-specific calcification in valvular tissue remain poorly understood. Here, we explored the role of osteopontin (OPN), a pro-fibrotic but anti-calcific bone sialoprotein, in regulating the calcification of female aortic valve tissue. Recognizing that OPN mediates calcification processes, we hypothesized that aortic valvular interstitial cells (VICs) in female tissue have reduced expression of osteogenic markers in the presence of elevated OPN relative to male VICs. Human female valve leaflets displayed reduced and smaller microcalcifications, but increased OPN expression relative to male leaflets. To understand how OPN expression contributes to observed sex dimorphisms in valve tissue, we employed enzymatically degradable hydrogels as a 3D cell culture platform to recapitulate male or female VIC interactions with the extracellular matrix. Using this system, we recapitulated sex differences observed in human tissue, specifically demonstrating that female VICs exposed to calcifying medium have smaller mineral deposits within the hydrogel relative to male VICs. We identified a change in OPN dynamics in female VICs in the presence of calcification stimuli, where OPN deposition localized from the extracellular matrix to perinuclear regions. Additionally, exogenously delivered endothelin-1 to encapsulated VICs increased OPN gene expression in male cells, which resulted in reduced calcification. Collectively, our results suggest that increased OPN in female valve tissue may play a sex-specific role in mitigating mineralization during AVS progression.

Extracellular matrix stiffness controls cardiac valve myofibroblast activation through epigenetic remodeling.

Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS.

Hydrogel cultures reveal Transient Receptor Potential Vanilloid 4 regulation of myofibroblast activation and proliferation in valvular interstitial cells.

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Impact of Collagen Triple Helix Structure on Melanoma Cell Invadopodia Formation and Matrix Degradation upon BRAF Inhibitor Treatment.

A collagen-rich tumor microenvironment (TME) is associated with worse outcomes in cancer patients and contributes to drug resistance in many cancer types. In melanoma, stiff and fibrillar collagen-abundant tissue is observed after failure of therapeutic treatments with BRAF inhibitors. Increased collagen in the TME can affect properties of the extracellular matrix (ECM), including stiffness, adhesiveness, and interaction of integrins with triple helix forming nanostructures. Decoupling these biochemical and biophysical properties of the ECM can lead to a better understanding of how each of these individual properties affect melanoma cancer behavior and drug efficacy. In addition, as drug treatment can induce cancer cell phenotypic switch, cancer cell responsiveness to the TME can be dynamically changed during therapeutic treatments. To investigate cancer cell phenotype changes and the role of the cancer TME, poly(ethylene glycol) (PEG) hydrogels functionalized with collagen mimetic peptides (CMPs) is utilized, or an interpenetrating network (IPN) of type І collagen within the PEG system to culture various melanoma cell lines in the presence or absence of Vemurafenib (PLX4032) drug treatment is prepared. Additionally, the potential of using CMP functionalized PEG hydrogels, which can provide better tunability is explored, to replace the existing invadopodia assay platform based on fluorescent gelatin.

Matters of the heart: Cellular sex differences.

Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart's microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for considering cellular sex differences in the design of bioengineered in vitro disease models of the heart are provided.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-ß1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10 ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Collagen networks within 3D PEG hydrogels support valvular interstitial cell matrix mineralization.

Enzymatically degradable hydrogels were designed for the 3D culture of valvular interstitial cells (VICs), and through the incorporation of various functionalities, we aimed to investigate the role of the tissue microenvironment in promoting the osteogenic properties of VICs and matrix mineralization. Specifically, porcine VICs were encapsulated in a poly(ethylene glycol) hydrogel crosslinked with a matrix metalloproteinase (MMP)-degradable crosslinker (KCGPQG↓IWGQCK) and formed via a thiol-ene photoclick reaction in the presence or absence of collagen type I to promote matrix mineralization. VIC-laden hydrogels were treated with osteogenic medium for up to 15 days, and the osteogenic response was characterized by the expression of RUNX2 as an early marker of an osteoblast-like phenotype, osteocalcin (OCN) as a marker of a mature osteoblast-like phenotype, and vimentin (VIM) as a marker of the fibroblast phenotype. In addition, matrix mineralization was characterized histologically with Von Kossa stain for calcium phosphate. Osteogenic response was further characterized biochemically with calcium assays, and physically via optical density measurements. When the osteogenic medium was supplemented with calcium chloride, OCN expression was upregulated and mineralization was discernable at 12 days of culture. Finally, this platform was used to screen various drug therapeutics that were assessed for their efficacy in preventing mineralization using optical density as a higher throughput readout. Collectively, these results suggest that matrix composition has a key role in supporting mineralization deposition within diseased valve tissue.

Calcium Signaling Regulates Valvular Interstitial Cell Alignment and Myofibroblast Activation in Fast-Relaxing Boronate Hydrogels.

The role viscoelasticity in fibrotic disease progression is an emerging area of interest. Here, a fast-relaxing hydrogel system is exploited to investigate potential crosstalk between calcium signaling and mechanotransduction. Poly(ethylene glycol) (PEG) hydrogels containing boronate and triazole crosslinkers are synthesized, with varying ratios of boronate to triazole crosslinks to systematically vary the extent of stress relaxation. Valvular interstitial cells (VICs) encapsulated in hydrogels with the highest levels of stress relaxation (90%) exhibit a spread morphology by day 1 and are highly aligned (80 ± 2%) by day 5. Key myofibroblast markers, including α-smooth muscle actin (αSMA) and collagen 1a1 (COL1A1), are significantly elevated. VIC myofibroblast activation decreases by 42 ± 18% through inhibition of mechanotransduction, independently of VIC morphology and alignment. Calcium signaling through a transient receptor potential vanilloid 4 (TRPV4) is found to regulate VIC spreading, alignment, and activation in a time dependent manner. Inhibition of calcium signaling at early time points results in disturbed cell alignment, decreased mechanotransduction, and diminished activation, while inhibition at later time points only causes partially reduced myofibroblast activation. These results suggest a potential crosstalk mechanism, where calcium signaling acts upstream of mechanosensing and can regulate VIC myofibroblast activation independently of mechanotransduction.

Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells.

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1ß as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1ß) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.

Prospective Study of Epizootic Hemorrhagic Disease Virus and Bluetongue Virus Transmission in Captive Ruminants.

Bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) cause hemorrhagic disease (HD) in wild ruminants and bluetongue disease (BT) and epizootic hemorrhagic disease (EHD) in livestock. These viruses are transmitted by biting midges in the genus Culicoides (family Ceratopogonidae). Mortality from this disease can reach 90% in certain breeds of sheep and in white-tailed deer (Odocoileus virginianus). From January until December of 2012, we conducted a prospective study to determine the origin and routes of transmission of BTV and EHDV in captive deer and cattle. The objective was to determine the abundance of Culicoides spp. and BTV/EHDV infection prevalence in midges, cattle, and deer in an area experiencing an outbreak of BT and EHD. Agar gel immunodiffusion (AGID) tests to detect for EHDV and BTV antibodies were conducted on serum collected from cattle and deer, quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) was utilized for BTV/EHDV RNA detection in tissues from dead deer, and CDC miniature black light traps baited with dry ice were deployed to capture insects. The AGID results showed 19 out of 29 cattle and 18 out of 58 white-tailed deer seroconverted for these viruses during the vector season. Tradition gel-based reverse transcriptase polymerase chain reaction was utilized to determine serotype. Sixteen cows were positive for EHDV-2, EHDV-6, or BTV-12 and 15 deer positive for EHDV-1, EHDV-6, or BTV-12. Specimens from 14 species of Culicoides (Dptera: Ceratopogonidae) (Culicoides arboricola Root and Hoffman, Culicoides biguttatus Coquillett, Culicoides crepuscularis Malloch, Culicoides debilipalpis Lutz, Culicoides furens Poey, Culicoides haematopotus Malloch, Culicoides hinmani Khalaf, Culicoides nanus Root and Hoffman, Culicoides neopulicaris Wirth, Culicoides paraensis Goeldi, Culicoides stellifer Coquillet, Culicoides variipennis Coquillet, Culicoides villosipennis Root and Hoffman, and Culicoides venustus Hoffman) were captured and tested for BTV and EHDV using RT-qPCR assays. BTV viral nucleic acid was detected in three pools from three different species of midges: C. crepuscularis, C. debilipalpis, and C. stellifer.

Quantifying heart valve interstitial cell contractile state using highly tunable poly(ethylene glycol) hydrogels.

Valve interstitial cells (VIC) are the primary cell type residing within heart valve tissues. In many valve pathologies, VICs become activated and will subsequently profoundly remodel the valve tissue extracellular matrix (ECM). A primary indicator of VIC activation is the upregulation of α-smooth muscle actin (αSMA) stress fibers, which in turn increase VIC contractility. Thus, contractile state reflects VIC activation and ECM biosynthesis levels. In general, cell contraction studies have largely utilized two-dimensional substrates, which are a vastly different micro mechanical environment than 3D native leaflet tissue. To address this limitation, hydrogels have been a popular choice for studying cells in a three-dimensional environment due to their tunable properties and optical transparency, which allows for direct cell visualization. In the present study, we extended the use of hydrogels to study the active contractile behavior of VICs. Aortic VICs (AVIC) were encapsulated within poly(ethylene glycol) (PEG) hydrogels and were subjected to flexural-deformation tests to assess the state of AVIC contraction. Using a finite element model of the experimental setup, we determined the effective shear modulus µ of the constructs. An increase in µ resulting from AVIC active contraction was observed. Results further indicated that AVIC contraction had a more pronounced effect on µ in softer gels (72 ±â€¯21% increase in µ within 2.5 kPa gels) and was dependent upon the availability of adhesion sites (0.5-1 mM CRGDS). The transparency of the gel allowed us to image AVICs directly within the hydrogel, where we observed a time-dependent decrease in volume (time constant τ=3.04 min) when the AVICs were induced into a hypertensive state. Our results indicated that AVIC contraction was regulated by both the intrinsic (unseeded) gel stiffness and the CRGDS peptide concentrations. This finding suggests that AVIC contractile state can be profoundly modulated through their local micro environment using modifiable PEG gels in a 3D micromechanical-emulating environment. Moving forward, this approach has the potential to be used towards delineating normal and diseased VIC biomechanical properties using highly tunable PEG biomaterials. STATEMENT OF SIGNIFICANCE.

Improvements in Tritrichomonas foetus molecular testing.

Bovine trichomoniasis is a sexually transmitted disease that results in infertility, abortion, and calf age variability. To date, management strategies include testing for Tritrichomonas foetus and culling of infected males. Challenges associated with testing include cost of culture medium, time and labor burden of sample incubation and processing, and adverse effects of bacterial growth on detection sensitivity. To overcome these challenges, we developed a direct reverse-transcription quantitative real-time PCR (direct RT-qPCR) utilizing smegma, eliminating the use of culture medium. In an analysis of 166 field samples (56 positives and 110 negatives as determined using microscopic reading of cultures as the reference test), the direct RT-qPCR exhibited 100% diagnostic sensitivity and 100% specificity, whereas the currently employed qPCR (culture qPCR), which utilizes cultured samples, exhibited 95% diagnostic sensitivity and 100% specificity. Agreement between direct RT-qPCR and culture qPCR was 98%. Moreover, direct RT-qPCR identified 3 more positive samples and exhibited lower quantification cycle (Cq) values among positives by culture reading than did culture qPCR (direct RT-qPCR Cq range = 14.6-32.3 vs. culture qPCR Cq range = 18.7-37.4). The direct RT-qPCR enables simplified sample collection, elimination of culture medium, faster results, applicability in cows, and lower cost than culture qPCR.

FGF-2 inhibits contractile properties of valvular interstitial cell myofibroblasts encapsulated in 3D MMP-degradable hydrogels.

Valvular interstitial cells (VICs) are responsible for the maintenance of the extracellular matrix in heart valve leaflets and, in response to injury, activate from a quiescent fibroblast to a wound healing myofibroblast phenotype. Under normal conditions, myofibroblast activation is transient, but the chronic presence of activated VICs can lead to valve diseases, such as fibrotic aortic valve stenosis, for which non-surgical treatments remain elusive. We monitored the porcine VIC response to exogenously delivered fibroblast growth factor 2 (FGF-2; 100 ng/ml), transforming growth factor beta 1 (TGF-ß1; 5 ng/ml), or a combination of the two while cultured within 3D matrix metalloproteinase (MMP)-degradable 8-arm 40 kDa poly(ethylene glycol) hydrogels that mimic aspects of the aortic valve. Here, we aimed to investigate VIC myofibroblast activation and subsequent contraction or the reparative wound healing response. To this end, VIC morphology, proliferation, gene expression related to the myofibroblast phenotype [alpha smooth muscle actin (α-SMA) and connective tissue growth factor (CTGF)] and matrix remodeling [collagens (COL1A1 and COL3) and MMP1], and contraction assays were used to quantify the cell response. Treatment with FGF-2 resulted in increased cellular proliferation while reducing the myofibroblast phenotype, as seen by decreased expression of CTGF and α-SMA, and reduced contraction relative to untreated control, suggesting that FGF-2 encourages a reparative phenotype, even in the presence of TGF-ß1. TGF-ß1 treatment predictably led to an increased proportion of VICs exhibiting the myofibroblast phenotype, indicated by the presence of α-SMA, increased gene expression indicative of matrix remodeling, and bulk contraction of the hydrogels. Functional contraction assays and biomechanical analyses were performed on VIC encapsulated hydrogels and porcine aortic valve tissue explants to validate these findings.

Three-Dimensional High-Throughput Cell Encapsulation Platform to Study Changes in Cell-Matrix Interactions.

In their native extracellular microenvironment, cells respond to a complex array of biochemical and mechanical cues that can vary in both time and space. High-throughput methods that allow characterization of cell-laden matrices are valuable tools to screen through many combinations of variables, ultimately helping to evolve and test hypotheses related to cell-ECM signaling. Here, we developed a platform for high-throughput encapsulation of cells in peptide-functionalized poly(ethylene glycol) hydrogels. Hydrogels were synthesized using a thiol-ene, photoclick reaction, which allowed the cell matrix environment to be modified in real time. Matrix signals were dynamically altered by in situ tethering of RGDS (0-1.5 mM), a fibronectin-derived adhesive peptide that induced more elongation than RLD or IKVAV, and/or by increasing the matrix modulus (1 to 6 kPa). This method was demonstrated with aortic valvular interstitial cells (VICs), a population of cells responsible for the pathological fibrosis and matrix remodeling that leads to aortic stenosis. VIC response to cell-matrix interactions was characterized by quantifying cell morphology and the fraction of cells exhibiting α-smooth muscle actin (αSMA) stress fibers, a hallmark of the myofibroblast phenotype. VICs elongated in response to RGDS addition, with a dramatic change in morphology within 24 h. Myofibroblast activation was also dependent on RGDS addition, with VICs exhibiting high activation (16-24%) in 1 kPa gels with RGDS. Response to RGDS was path-dependent, with the amount of time exposed to the adhesive ligand important in determining VIC morphology and activation. Although VIC aspect ratios were dependent on the amount of time spent in a stiff vs soft gel, low levels of VIC activation (≤4%) were observed in any gels cultured in higher modulus (6 kPa vs 1 kPa) microenvironments.

Molecular and serological in-herd prevalence of Anaplasma marginale infection in Texas cattle.

Bovine anaplasmosis is an infectious, non-contagious disease caused by the rickettsial pathogen Anaplasma marginale (A. marginale). The organism has a global distribution and infects erythrocytes, resulting in anemia, jaundice, fever, abortions and death. Once infected, animals remain carriers for life. The carrier status provides immunity to clinical disease, but is problematic if infected and naïve cattle are comingled. Knowledge of infection prevalence and spatial distribution is important in disease management. The objective of this study was to assess A. marginale infection in-herd prevalence in Texas cattle using both molecular and serological methods. Blood samples from 11 cattle herds within Texas were collected and analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) and a commercial competitive enzyme-linked immunosorbent assay (cELISA). Samples from experimentally infected animals were also analyzed and RT-qPCR detected A. marginale infection up to 15 days before cELISA, providing empirical data to support the interpretation of herd prevalence results. Herds with high prevalence were located in the north Texas Rolling Plains and west Trans-Pecos Desert, with RT-qPCR prevalence as high as 82% and cELISA prevalence as high as 88%. Overall prevalence was significantly higher in cattle in north and west Texas compared to cattle in east Texas (p<0.0001 for prevalence based on both RT-qPCR and cELISA). The overall RT-qPCR and cELISA results exhibited 90% agreement (kappa=0.79) and provide the first A. marginale infection prevalence study for Texas cattle using two diagnostic methods. Since cattle are the most important reservoir host for A. marginale and can serve as a source of infection for tick and mechanical transmission, information on infection prevalence is beneficial in the development of prevention and control strategies.

Detection of African swine fever, classical swine fever, and foot-and-mouth disease viruses in swine oral fluids by multiplex reverse transcription real-time polymerase chain reaction.

African swine fever (ASF), classical swine fever (CSF), and foot-and-mouth disease (FMD) are highly contagious animal diseases of significant economic importance. Pigs infected with ASF and CSF viruses (ASFV and CSFV) develop clinical signs that may be indistinguishable from other diseases. Likewise, various causes of vesicular disease can mimic clinical signs caused by the FMD virus (FMDV). Early detection is critical to limiting the impact and spread of these disease outbreaks, and the ability to perform herd-level surveillance for all 3 diseases rapidly and cost effectively using a single diagnostic sample and test is highly desirable. This study assessed the feasibility of simultaneous ASFV, CSFV, and FMDV detection by multiplex reverse transcription real-time polymerase chain reaction (mRT-qPCR) in swine oral fluids collected through the use of chewing ropes. Animal groups were experimentally infected independently with each virus, observed for clinical signs, and oral fluids collected and tested throughout the course of infection. All animal groups chewed on the ropes readily before and after onset of clinical signs and before onset of lameness or serious clinical signs. ASFV was detected as early as 3 days postinoculation (dpi), 2-3 days before onset of clinical disease; CSFV was detected at 5 dpi, coincident with onset of clinical disease; and FMDV was detected as early as 1 dpi, 1 day before the onset of clinical disease. Equivalent results were observed in 4 independent studies and demonstrate the feasibility of oral fluids and mRT-qPCR for surveillance of ASF, CSF, and FMD in swine populations.