Bovine Red Blood Cells Research Articles

Risk of BSE transmission when fishmeal derived from fish fed bovine spray-dried red blood cells is included in calf milk replacers

The use of residual streams from agricultural production and food consumption containing animal proteins entails the risk of disease transmission as illustrated by the epidemics of bovine spongiform encephalopathy (BSE) and African swine fever. To combat this risk, the use of animal proteins in livestock feed was banned in the European Union, resulting in a drain of valuable proteins from the agricultural system. With an increasing call for a circular food system, the use of residual streams as a feed ingredient needs to be reconsidered with the associated disease risks being assessed and mitigated where needed. In this study, we assessed the BSE risk of bovine spray-dried red blood cells (SDRBC) as an ingredient of aquafeed. Fish fed with bovine SDRBC could indirectly result in exposure of ruminants to BSE infectivity because one of the exemptions of the feed ban is the use of fishmeal as an ingredient in calf milk replacers. A quantitative risk model was built to evaluate the BSE infectivity present in blood sourced from a slaughtered BSE-infected cow and the reduction of infectivity due to processing steps along the production chain. The end point of the model was the BSE infectivity, expressed in cattle oral ID50 (CoID50), reaching calves fed calf milk replacer containing fishmeal, and the corresponding probability that this will result in at least one new BSE infection.The expected BSE infectivity in blood from a BSE-infected cow at the clinical end state of infection is 0.75 CoID50 (median value). Infectivity in blood mainly results from cross-contamination with brain tissue during stunning at the slaughterhouse. The initial infectivity is reduced along the pathway from slaughtered cow to calf milk replacer, with the highest reduction achieved by clearance of infectivity by fish fed bovine SDRBC as an ingredient of aquafeed, although this parameter has high uncertainty. The final infectivity reaching calves via inclusion of fishmeal in calf milk replacer is estimated to be very low (median value: 1.1 × 10−5 CoID50). Assuming an exponential dose-response model, this corresponds with an expected probability that < 10 out of a million slaughtered BSE-infected cows will result in new BSE infections, which is far below the threshold value of 1 for the basic reproduction number (R0) to initiate a new epidemic. We thus conclude that it is very unlikely that the use of bovine SDRBC as ingredient of aquafeed will result in a new BSE epidemic in cattle. What-if analysis indicated that this conclusion is robust, despite high uncertainty for some input parameters.

Comparative Evaluation of UV-Vis Spectroscopy-Based Approaches for Hemoglobin Quantification: Method Selection and Practical Insights.

The growing demand for effective alternatives to red blood cells (RBCs) has spurred significant research into hemoglobin (Hb)-based oxygen carriers (HBOCs). Accurate characterization of HBOCs-including Hb content, encapsulation efficiency, and yield-is crucial for ensuring effective oxygen delivery, economic viability, and the prevention of adverse effects caused by free Hb. However, the choice of quantification methods for HBOCs is often driven more by tradition than by a thorough assessment of available options. This study meticulously compares various UV-vis spectroscopy-based methods for Hb quantification, focusing on their efficacy in measuring Hb extracted from bovine RBCs across different concentration levels. The findings identify the sodium lauryl sulfate Hb method as the preferred choice due to its specificity, ease of use, cost-effectiveness, and safety, particularly when compared to cyanmethemoglobin-based methods. Additionally, the study discusses the suitability of these methods for HBOC characterization, emphasizing the importance of considering carrier components and potential interferences by analyzing the absorbance spectrum before selecting a method. Overall, this study provides valuable insights into the selection of accurate and reliable Hb quantification methods, which are essential for rigorous HBOC characterization and advancements in medical research.

Identification of Anaplasma marginale adhesins for bovine erythrocytes using phage display

Bovine anaplasmosis, caused by Anaplasma marginale, is one of the most common tick-borne diseases of cattle and has a worldwide distribution. The high costs of bovine anaplasmosis are due to treatment, decreased production, and outbreaks resulting in high mortality. The impact of bovine anaplasmosis is greatest in tropical and subtropical regions where tick vectors are abundant year around. Prevention generally relies on the use of tetracyclines to prevent disease and acaricides to reduce tick burdens. Thus, additional methods to prevent disease while reducing the use of antibiotics are needed. Protection can be reliably achieved with immunization using outer membrane proteins, thus allowing for the possibility for development of a recombinant vaccine. However, prioritizing the selection and testing of antigens from the protective outer membrane extract remains a challenge. Because A. marginale is an obligate intracellular pathogen, surface proteins that mediate adhesion to host cells, primarily red blood cells (RBCs), are functionally relevant vaccine candidates. With some exceptions, the proteins that bind RBCs remain unknown. To address this gap, a phage display library expressing 66 A. marginale proteins was screened to identify adhesins for bovine RBCs. Of the screened proteins, 73% were eliminated due to poor binding to RBCs. Several potential adhesins were identified, including Msp1b and OmpA, which are known adhesions for bovine RBCs and tick cells, respectively. Additionally, Mlp3, Am779, Msp3, and Omp13 met the criteria as RBCs adhesins and may serve as high priority vaccine candidates for future testing.

The Identification of a Novel Spider Toxin Peptide, Lycotoxin-Pa2a, with Antibacterial and Anti-Inflammatory Activities.

With the increasing challenge of controlling infectious diseases due to the emergence of antibiotic-resistant strains, the importance of discovering new antimicrobial agents is rapidly increasing. Animal venoms contain a variety of functional peptides, making them a promising platform for pharmaceutical development. In this study, a novel toxin peptide with antibacterial and anti-inflammatory activities was discovered from the spider venom gland transcriptome by implementing computational approaches. Lycotoxin-Pa2a (Lytx-Pa2a) showed homology to known-spider toxin, where functional prediction indicated the potential of both antibacterial and anti-inflammatory peptides without hemolytic activity. The colony-forming assay and minimum inhibitory concentration test showed that Lytx-Pa2a exhibited comparable or stronger antibacterial activity against pathogenic strains than melittin. Following mechanistic studies revealed that Lytx-Pa2a disrupts both cytoplasmic and outer membranes of bacteria while simultaneously inducing the accumulation of reactive oxygen species. The peptide exerted no significant toxicity when treated to human primary cells, murine macrophages, and bovine red blood cells. Moreover, Lytx-Pa2a alleviated lipopolysaccharide-induced inflammation in mouse macrophages by suppressing the expression of inflammatory mediators. These findings not only suggested that Lytx-Pa2a with dual activity can be utilized as a new antimicrobial agent for infectious diseases but also demonstrated the implementation of in silico methods for discovering a novel functional peptide, which may enhance the future utilization of biological resources.

Examination of Bovine Red Blood Cell Death in Vitro in Response to Pathophysiologic Proapoptotic Stimuli.

Interspecies variations in mammalian red blood cells (RBCs) are observed in circulating RBC lifespan, cell size, fluidity, aggregation, water permeability, metabolism, lipid composition, and the overall proteome. Bovine RBC cell membrane is deficient in phosphatidylcholine and exhibits anomalies in the arrangement of phosphatidylethanolamine within the lipid bilayer. However, like human RBCs, virtually all the aminophospholipid phosphatidylserine (PS) is found within the cytoplasmic side of the cell membrane of intact circulating bovine RBCs. During apoptotic cell death of human and murine RBCs, PS translocates to the outer leaflet of the cell membrane via Ca2+-dependent and -independent signaling mechanisms. However, little is known about this process in bovine RBCs. Using cytofluorometry analyses, we characterized and compared the cell death responses in bovine and human RBCs in vitro exposed to various pathophysiologic cell stressors. Ionic stress, by ionophore treatment, and oxidative stress enhanced cytoplasmic Ca2+ levels and cell membrane PS expression in both bovine and human RBCs. Fever-grade hyperthermia and energy starvation promoted Ca2+ influx and elevated reactive oxygen species levels in both human and bovine RBCs. However, bovine RBCs displayed minimal increases in PS expression elicited by hyperthermia, energy starvation, and extracellular hypertonicity as compared to human RBCs. In response to decreased extracellular osmolality, bovine RBCs exhibited significantly enhanced fragility as compared to human RBCs. Bovine RBCs display differential cell death patterns as compared to human RBCs, only partly explained by increased Ca2+ influx and oxidative stress. Premature removal of circulating RBCs could potentially contribute to the pathogenesis of anemia in cattle caused by a wide range of factors such as systemic diseases, parasitic infections, and nutritional deficiencies.

Changes in the sensitivity of mammalian erythrocytes to hypertonic shock and cryohemolysis under the pretreatment by phenylhydrazine

The effect of pretreating mammalian erythrocytes with phenylhydrazine on their sensitivity to hypertonic shock and hypertonic cryohemolysis was investigated. The results of the experiments showed that the sensitivity of intact mammalian erythrocytes to these stress effects is species-specific. It can be determined by differences in the protein and phospholipid composition of the erythrocytes studied. Human erythrocytes are more sensitive to hypertonic shock at 37 and 0°C, and human and equine erythrocytes are more sensitive to hypertonic cryohemolysis. It was found that under hypertonic shock conditions, the degree of lysis of rabbit erythrocytes at 37°C and 0°C is the same, whereas that of bovine red blood cells is significantly different. Phenylhydrazine treatment alters the sensitivity of erythrocytes to hypertonic shock of some studied mammals and to hypertonic cryohemolysis in all of them. The results showed that under hypertonic shock at 37°C, the sensitivity of human and bovine cells decreases, that of rabbit cells does not change, that of horse cells increases; at 0°C, it increases in all species studied. It should be noted that the sensitivity of horse erythrocytes to hypertonic injury increases significantly (almost twice) at 0 and 37°C, whereas the sensitivity of rabbit erythrocytes does not change at 37°C. Under conditions of hypertonic cryohemolysis, the degree of cell lysis after treatment with phenylhydrazine becomes the same for erythrocytes of all mammalian species studied, i.e. the effect of stress becomes universal and not species-specific. Taking into account the data on the effect of phenylhydrazine only on the protein part of the erythrocyte cytoskeleton-membrane complex, it can be assumed that the protein component of the cytoskeleton is decisive in the response of mammalian erythrocytes to the effect of hypertonic cryohemolysis. As for hypertonic shock, since the species-specificity of the mammalian erythrocyte response to stress is preserved after phenylhydrazine action on membrane proteins, other structures, such as the lipid component of the membrane, could determine the sensitivity of erythrocytes to this type of stress.

Disruption of a DNA fragment that encodes the microneme adhesive repeat domain-containing region of the BBOV_III011730 does not affect the blood stage growth of Babesia bovis in vitro

Babesia bovis, an intraerythrocytic hemoprotozoan parasite, causes the most pathogenic form of bovine babesiosis, negatively impacting the cattle industry. Comprehensive knowledge of B. bovis biology is necessary for developing control methods. In cattle, B. bovis invades the red blood cells (RBCs) and reproduces asexually. Micronemal proteins, which bind to sialic acid of host cells via their microneme adhesive repeat (MAR) domains, are believed to play a key role in host cell invasion by apicomplexan parasites. In this study, we successfully deleted the region encoding MAR domain of the BBOV_III011730 by integrating a fusion gene of enhanced green fluorescent protein-blasticidin-S-deaminase into the genome of B. bovis. The transgenic B. bovis, lacking the MAR domain of the BBOV_III011730, invaded bovine RBCs in vitro and grew at rates similar to the parental line. In conclusion, our study revealed that the MAR domain is non-essential for the intraerythrocytic development of B. bovis in vitro.

Bioinspired Polycation Functionalization of the Polyurethane Surface for Enhanced Lubrication, Antibacterial Property, and Anticoagulation

Endowing the implanted biomaterial with a wide range of functions, such as lubrication and antibacterial and anticoagulation properties, is essential for their safe clinical use. In the present study, based on the superlubrication mechanism of articular cartilage, a branched polyelectrolyte polymer (PEI-PMPC) was successfully synthesized based on tert-butyl hydroperoxide-initiated grafting polymerization and then applied to the biocompatible polyurethane (PU) surface. Specifically, the PU sheet was pretreated by polydopamine (PDA), and the PEI-PMPC polymer was grafted onto the PDA-modified PU surface by Schiff base reaction to generate a solid PDA/PEI-PMPC coating. The unmodified and modified surfaces were characterized by Fourier transform infrared spectroscopy, water contact angle, X-ray photoelectron spectroscopy, and surface zeta potential. To compare the lubrication, antibacterial behavior, and hemocompatibility, the relevant properties of the modified surfaces were evaluated by the tribological test, plate count method, surface morphology, hemolysis rate, activated partial thromboplastin time, and thrombin time. The experimental results demonstrated that the adhesion of bovine serum albumin, Escherichia coli, Staphylococcus aureus, platelets, and red blood cells was significantly decreased on the PEI-PMPC-modified PU surface compared with that of the unmodified PU surface. The multi-functional properties of the coating were attributed to the hydration layer formed by zwitterionic phosphorylcholine groups, and additionally, the cationic PEI served to kill the bacteria adhering to the surface. In summary, the bioinspired coating proposed in this study could potentially be used as a promising surface functionalization strategy for biomedical implants.

A small-angle X-ray scattering study of red blood cells in continuous flow.

Owing to their large penetration depth and high resolution, X-rays are ideally suited to study structures and structural changes within intact biological cells. For this reason, X-ray-based techniques have been used to investigate adhesive cells on solid supports. However, these techniques cannot easily be transferred to the investigation of suspended cells in flow. Here, an X-ray compatible microfluidic device that serves as a sample delivery system and measurement environment for such studies is presented. As a proof of concept, the microfluidic device is applied to investigate chemically fixed bovine red blood cells by small-angle X-ray scattering (SAXS). A very good agreement is found between in-flow and static SAXS data. Moreover, the data are fitted with a hard-sphere model and screened Coulomb interactions to obtain the radius of the protein hemoglobin within the cells. Thus, the utility of this device for studying suspended cells with SAXS in continuous flow is demonstrated.

Blood Cell Separation Using Polypropylene-Based Microfluidic Devices Based on Deterministic Lateral Displacement.

Mammalian blood cell separation methods contribute to improving the diagnosis and treatment of animal and human diseases. Microfluidic deterministic lateral displacement (DLD) devices can sort cells based on their particle diameter. We developed microfluidic DLD devices with poly(propylene)-based resin and used them to separate bovine and human red blood cells (RBCs) and white blood cells (WBCs) without electric devices. To determine the critical cut-off diameter (Dc) of these devices, we used immunobeads with a diameter of 1-20 μm. The Dc values of the microfluidic DLD devices for the immunobeads in the experiments were similar to the calculated Dc values (8-10 μm). Results from bovine blood cell separation experiments suggest that lymphocytes and neutrophils can be separated from diluted, whole blood. Human RBCs were occasionally observed in the left outlet where larger particles with diameters closer to the Dc value were collected. Based on the Dc values, human neutrophils were sorted to the left outlet, whereas lymphocytes were observed in both outlets. Although microfluidic channel optimization is required for the concentration of sorted cells, the microfluidic DLD device prepared with a poly(propylene)-based resin has the potential for clinical use.

Evaluation of Senna tora (L.) Roxb. leaves as source of bioactive molecules with antioxidant, anti-inflammatory and antibacterial potential

Senna tora (L.) Roxb. is an ethno-medicinal herb used by rural and tribal people of the Satpura region of Madhya Pradesh in India and the Phatthalung Province of Thailand for treating rheumatism, bronchitis, ringworm, itches, leprosy, dyspepsia, liver disorders and heart disorders. It is also used in Chinese and Ayurvedic medicine. This study was conducted to investigate the potential of Senna tora (L.) Roxb. as a source of drug candidates against oxidants, inflammation, and bacterial infection. Preliminary phytochemical screening (PPS) and GC-MS were performed to identify the phytochemicals in the ethyl acetate extract of Senna tora (L.) Roxb. leaves (EAESTL). The in vitro antioxidant activity was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH)- and H2O2-scavenging tests; the in vitro anti-inflammatory activity was determined by bovine serum albumin (BSA) denaturation and red blood cell (RBC) hemolysis inhibition; and the antibacterial activity was evaluated by agar-well diffusion methods. Cytotoxicity was estimated by Artemia salina larvae lethality, while acute toxicity was evaluated by oral delivery of the extract to mice. In silico antioxidant, anti-inflammatory, and antibacterial activities were predicted by the Prediction of Activity Spectra for Substances (PASS) program. The pharmacokinetics related to ADME and toxicity tests were determined by the admetSAR2 and ADMETlab2 web servers, and drug-able properties were assessed by the SwissADME server. GC-MS detected fifty-nine phytochemicals that support the types of compounds (phenols, flavonoids, tannins, terpenoids, saponins, steroids, alkaloids, glycosides and reducing sugar) identified by phytochemical screening. EAESTL exhibited dose-dependent antioxidant, anti-inflammatory, and antibacterial activities without any adverse effects or fluctuations in body weight. The PASS program predicted that the identified phytochemicals have antioxidant, anti-inflammatory and antibacterial activities. Among 51 phytochemicals, 16 showed good ADME, and 8 fulfilled drug-able properties without toxicity. Altogether, four phytochemicals, viz., benzyl alcohol, 3-(hydroxy-phenyl-methyl)-2,3-dimethyl-octan-4-one, phenylethyl alcohol and 2,6,6-trimethylbicyclo [3.1.1] heptane-3-ol, showed good pharmacokinetics and drug-able properties without toxicity, along with antioxidant, anti-inflammatory, and antibacterial activities. The obtained results suggest that Senna tora (L.) Roxb. leaves contain bioactive phytochemicals that have the potential to fight against oxidants, inflammation, and bacterial infection as potential drug candidates.

Examining the universality of the hemolysis power law model from simulations of the FDA nozzle using calibrated model coefficients.

Computational fluid dynamics (CFD) is widely used to predict mechanical hemolysis in medical devices. The most popular hemolysis model is the stress-based power law model that is based on an empirical correlation between hemoglobin release from red blood cells (RBCs) and the magnitude of flow-induced stress and exposure time. Empirical coefficients are traditionally calibrated using data from experiments in simplified Couette-type blood-shearing devices with uniform-shear laminar flow and well-defined exposure times. Use of such idealized coefficients in simulations of real medical devices with complex hemodynamics is thought to be a primary reason for the historical inaccuracy of absolute hemolysis predictions using the power law model. Craven et al.(Biomech Model Mechanobiol 18:1005-1030, 2019) recently developed a CFD-based Kriging surrogate modeling approach for calibrating empirical coefficients in real devices that could potentially be used to more accurately predict absolute hemolysis. In this study, we use the FDA benchmark nozzle to investigate whether utilizing such calibrated coefficients improves the predictive accuracy of the standard Eulerian power law model. We first demonstrate the credibility of our CFD flow simulations by comparing with particle image velocimetry measurements. We then perform hemolysis simulations and compare the results with in vitro experiments. Importantly, the simulations use coefficients calibrated for the flow of a suspension of bovine RBCs through a small capillary tube, which is relatively comparable to the flow of bovine blood through the FDA nozzle. The results show that the CFD predictions of relative hemolysis in the FDA nozzle are reasonably accurate. The absolute predictions are, however, highly inaccurate with modified index of hemolysis values from CFD in error by roughly three orders of magnitude compared with the experiments, despite using calibrated model coefficients from a relatively similar geometry. We rigorously examine the reasons for the inaccuracy that include differences in the flow conditions in the hemolytic regions of each device and the lack of universality of the hemolysis power law model that is entirely empirical. Thus, while the capability to predict relative hemolysis is valuable for product development, further improvements are needed before the power law model can be relied upon to accurately predict the absolute hemolytic potential of a medical device.

Identification of novel immune correlates of protection against acute bovine babesiosis by superinfecting cattle with in vitro culture attenuated and virulent Babesia bovis strains.

The apicomplexan tickborne parasites Babesia bovis and B. bigemina are the major causative agents of bovine babesiosis, a disease that negatively affects the cattle industry and food safety around the world. The absence of correlates of protection represents one major impediment for the development of effective and sustainable vaccines against bovine babesiosis. Herein we superinfected cattle with attenuated and virulent strains of B. bovis to investigate immune correlates of protection against acute bovine babesiosis. Three 6-month-old Holstein calves were infected intravenously (IV) with the in vitro culture attenuated Att-S74-T3Bo B. bovis strain (106 infected bovine red blood cells (iRBC)/calf) while three age-matched Holstein calves were inoculated IV with normal RBC as controls (106 RBC/calf). All Att-S74-T3Bo-infected calves showed a significant increase in temperature early after inoculation but recovered without treatment. Att-S74-T3Bo-infected calves also developed: (a) monocytosis, neutropenia, and CD4+ lymphopenia in peripheral blood on days 3 to 7 post-inoculation; (b) significant levels of TNFα, CXCL10, IFNγ, IL-4, and IL-10 in sera at day 6 after infection; and (c) IgM and IgG against B. bovis antigens, starting at days 10 and 30 post-inoculation, respectively. At 46 days post-Att-S74-T3Bo inoculation, all experimental calves were infected IV with the homologous virulent B. bovis strain Vir-S74-T3Bo (107 iRBC/calf). All Att-S74-T3Bo-infected calves survived superinfection with Vir-S74-T3Bo without displaying signs of acute babesiosis. In contrast, control animals showed signs of acute disease, starting at day 10 post-Vir-S74-T3Bo infection, and two of them were humanely euthanized at days 13 and 14 after inoculation due to the severity of their symptoms. Also, control calves showed higher (P<0.05) parasite load in peripheral blood compared to animals previously exposed to Att-S74-T3Bo. No significant alterations in the profile of leukocytes and cytokines were observed in Att-S74-T3Bo-inoculated after Vir-S74-T3Bo infection. In conclusion, data demonstrate novel changes in the profile of blood immune cells and cytokine expression in peripheral blood that are associated with protection against acute bovine babesiosis. These identified immune correlates of protection may be useful for designing effective and sustainable vaccines against babesiosis in cattle.

Bovine erythrocytes are poor surrogates for human when exposed to sublethal shear stress.

Animal blood products are routinely used as surrogates for human tissue in haemocompatibility testing of rotary blood pumps. Bovine blood is particularly attractive due to the animal's large blood volume; however, bovine red blood cells (RBC) differ substantially from those of human, both in biophysical properties and molecular composition. We aimed to determine whether differences also exist in the sensitivity of bovine RBC to a standardised shear stress protocol. Fresh blood from healthy human and bovine donors was exposed to discrete combinations of shear stress using a Couette shearing system, prior to assessment of cellular deformability and mechanical sensitivity. Each sample was exposed to 25 sublethal shear stress combinations (ranging 60-100 Pa × 5-300 s). While bovine RBC exhibited decreased maximal elongation in the absence of conditioning shear, overall deformability at lower shears was ~1.8-fold greater than human. When exposed to any conditioning shear stresses >80 Pa (or 60-70 Pa beyond 5 s), human RBC were significantly rigidified, with greater magnitudes and prolonged exposure compounding this effect. Significantly larger shears were required to rigidify bovine RBC; the most extreme shear condition (100 Pa × 300 s) resulted in approximately three-times more rigidification of human RBC than bovine (137% and 47% respectively). Bovine RBC have superior resilience to mechanical stress when compared with human. Using bovine blood in ex vivo evaluation of rotary blood pumps may thus misrepresent and overestimate device-blood success, and may also have flow-on effects for eventual users. Fresh human blood during early-phase ex vivo testing is thus recommended, given shear-inducing blood pumps are designed for humans - not cattle.

Comparative evaluation of cadmium-induced oxidative stress in camel and bovine erythrocytes

ObjectivesErythrocytes or red blood cells (RBCs) are the most abundant cells in blood. They transport oxygen from lungs to tissues and bring back carbon dioxide from tissues to lungs. Being non-nucleated and lacking the cellular organelles, they have been widely used for in vitro toxicity studies. The present study was carried out to study the comparative in vitro toxicity of cadmium (Cd) on RBCs of camel and cow. MethodsBoth cow and camel whole blood samples were exposed to 0, 10, 50 and 100 μM of cadmium chloride (CdCl2) and the tubes were incubated at 37 °C for 24 h. The erythrocytes from both the species were separated and used for biochemical determinations for markers of oxidative stress. ResultsCd caused a concentration dependent decrease in bovine glutathione (GSH) content and superoxide dismutase (SOD) activity. However, in the case of camel RBCs, the decrease in GSH content and SOD activity were observed at medium and high concentrations only. Cd exposure also resulted in significant inhibition of glutathione peroxidase (GPx) and glutathione S-transferase in both bovine and camel RBCs. The GPx in camel RBCs was inhibited only at a high concentration of Cd. Various concentrations of Cd inhibited the catalase in bovine RBCs only, without any significant inhibition of catalase in camel RBCs. Medium and high concentrations of Cd caused an increase in lipid peroxidation in bovine RBCs. In the case of camel RBCs, none of the used concentrations of Cd caused lipid peroxidation. ConclusionsThe present study concludes that in vitro incubation of camel and bovine RBCs with Cd causes an impairment of their antioxidant system and therefore increases their susceptibility to hemolysis. However, the camel RBCs appear to have an increased resistance to oxidative stress generated by cadmium exposure.

Assessment of the hemagglutinating activity of the Porcine orthorubulavirus

Blue eye disease (BED) in pigs is caused by Porcine orthorubulavirus (PRV) of the Paramyxoviridae family. It is an endemic disease in swine production in the central region of Mexico and causes nervous signs and high mortality in suckling pigs, pneumonia in growing pigs, orchitis in boars and mummification during gestation. PRV hemagglutinates most red blood cells (RBCs) of domestic species. For serological diagnosis, the hemagglutination inhibition test is used, and in this test, guinea pig, bovine and chicken RBCs have been commonly used. In this investigation, hemagglutination with PRV was evaluated using the RBCs of seven domestic species (chicken, bovine, horse, pig, dog, guinea pig and rabbit). In the hemagglutination test, the following parameters were evaluated: temperature (25 °C and 37 °C), bottoms of the wells (V and U), erythrocyte concentration (0.5%, 0.75%, and 1%), and reading time (15, 30, 45, 60 and 90 min). Significant differences (P < 0.001) were found in most of the evaluated treatments. The best hemagglutination results were obtained with chicken, bovine and horse RBCs. The hemagglutination titer is higher (2 dilutions) when using chicken RBCs than when using bovine or horse RBCs. If chicken RBCs are used in the inhibition of hemagglutination, the test will be more sensitive, while it is more specific when bovine or horse RBCs are used. The hemagglutination readings are imprecise when using RBCs from dogs, pigs, guinea pigs and rabbits. RBCs from these species should not be used for the diagnosis or investigation of PRV.

Hemorheological Approach to Improve Perfusion of Red Blood Cells with Reduced Deformability Using Drag-Reducing Polymer (In Vitro Study).

Drag-reducing polymers (DRPs) are nontoxic water-soluble blood additives that have been shown to beneficially alter hemodynamics when delivered intravenously in nanomolar concentrations. This study examines the ability of DRPs to alter the traffic of mixtures of normal and less-deformable red blood cells (RBCs) through branched microchannels and is intended to support and expand upon previous experiments within straight capillary tubes to promote DRPs for future clinical use. Branched polydimethylsiloxane microchannels were perfused with a mixture of normal bovine RBCs also containing heat-treated less-deformable RBCs at a hematocrit of 30% with 10 ppm of the DRP poly(ethylene oxide) (MW 4M Da). Suspensions were driven by syringe pump, collected at outlets, and RBC dimensions measured while subject to shear stress to determine the proportion of healthy RBCs in each sample. DRPs eliminated evidence of the plasma skimming phenomena and significantly increased the pressure drop across microchannels. Further, DRPs were found to cause an increase in the proportion of healthy RBCs exiting the branch outlet from -8.5 ± 2.5% (control groups) to +12.1 ± 5.4% (n = 6, p = 0.02). These results suggest DRP additives may be used to improve the perfusion of less-deformable RBCs in vivo and indicates their potential for future clinical use.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Correlation of Oxygen‐offloading rate from Red Blood Cells with Body Mass

The diffusion of O2 across red blood cell (RBC) membranes is critically important for the delivery of O2 to peripheral tissues. Although the traditional view had been that all O2 moves through RBC membranes by dissolving in and diffusing through membrane lipid, our recent work on murine RBCs indicates that most O2 traversing RBC membranes moves via protein channels (bioRxiv. doi:10.1101/2020.08.28.265066). In stopped-flow (SF) kinetics experiments, we use the absorbance spectrum of hemoglobin (Hb) to monitor the rate constant for O2 offloading from Hb (kHbO2). In RBCs from mice genetically deficient in both aquaporin-1 (AQP1) and the Rh complex (mainly RhAG), kHbO2 is 1/3 lower than in RBCs from wild-type (WT) mice. According to mathematical simulations, this 1/3 decrease in kHbO2 corresponds to a ~55% decrease in O2 permeability of RBC membranes (PM,O2). The combination of the double-knockout and pCMBS (nonspecific inhibitor of membrane proteins that is nonetheless excluded from the RBC interior) reduces PM,O2 by ~91%. Control experiments—hematology, RBC morphology, proteomics (abundance of individual proteins), lipidomics (abundance of individual membrane lipids)—rule out other potential explanations for our striking SF data. Our next challenge is to identify the membrane protein(s) responsible for the 91% – 55% = 36% of PM,O2 not accounted for by AQP1 and the Rh complex, as well as to establish the role of membrane lipids. Comparative physiology could be an important tool for achieving these goals. For each RBC sample, we correct the raw kHbO2 data for modest degrees of hemolysis that occur inside the SF reaction chamber. We assess this hemolysis using our novel SF assay, which is based on the release of carbonic anhydrase (CA) enzymes from RBCs. We find that, compared to RBCs from our lab-standard C57BL/6 WT mice, bovine RBCs have a significantly lower hemolysis (~2.5% vs. ~5%, N=3, p <0.01). More interestingly, we find a 71% lower CA activity in bovine RBCs compared to that in mouse RBCs (N=3, p <0.01). This observation could be important as we explore CO2 permeability in the near future. The kHbO2 is 41% lower for bovine RBCs than for RBCs from WT mice (N=3, p <0.01). Hematological analyses of our limited number of bovine samples do not yet show a significant difference from extensive murine samples in either mean corpuscular volume (MCV) or mean corpuscular hemoglobin concentration (MCHC). A further evaluation of hematology, morphology, proteomics, lipidomics, and genomics (for deduced amino-acid sequences of proteins), will provide valuable insight to the contribution of specific membrane proteins and lipids to the relatively low kHbO2 of bovine RBCs. Our comparative analysis would benefit greatly from access to other mammals with a wide range of body mass (e.g., up to elephant size), from which we would use a small amount of blood (<1 ml) in the assays described above.

Insight into Francisella tularensis-erythrocyte interactions

Francisella tularensis, the causative agent of tularemia, is a pathogenic gram-negative bacterium that enters human red blood cells (RBCs). Previous investigations suggest that invasion of RBCs helps F. tularensis survive in the gut of arthropods – a transmission vector of this bacterium leading to colonization. Since tularemia is a zoonotic infection, humans are not the primary host of F. tularensis. Therefore, we sought to investigate whether F. tularensis was capable of invading RBCs of other animals. Using the gentamicin protection assay, we discovered that while F. tularensis could enter sheep and chicken erythrocytes, bovine RBCs did not support invasion. Because of previous studies published by our lab, and due to intrinsic differences between human and bovine erythrocyte surface protein Band 3, we hypothesized that RBC membrane protein Band 3 is required for invasion. We used anti-band 3 Fab fragments to physically block the Band 3 RBC surface protein and investigated how that impacted F. tularensis invasion. Here, we provide preliminary evidence suggesting that F. tularensis uses Band 3 to enter human RBCs. This study was funded by the National Institute of Allergy and Infectious Diseases (1R15HL147135) and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health (P20GM103434), which funds WV-INBRE program.