Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of African Horse Sickness Virus.

African horse sickness (AHS) is a disease of equids caused by African Horse Sickness Virus (AHSV) and is transmitted by Culicoides midges. AHS is endemic in sub-Saharan Africa, but during the past century, outbreaks of significant economic importance and elevated mortality have been recorded in Northern African countries, the Iberian and Arabian Peninsula, the Middle East and the Indian subcontinent. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Conventional reverse-transcriptase (RT) PCR (RT-PCR) and real-time RT-PCR (rRT-PCR) assays have improved the sensitivity and rapidity of diagnosing AHS, resulting in the adoption of these methods as recommended tests by the World Organisation for Animal Health (OIE). However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for AHS would improve the fast implementation of control policies. Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement nucleic acid amplification technique which can be performed in the field. LAMP assays are attractive molecular assays because they are simple to use, rapid, portable and have sensitivity and specificity within the range of rRT-PCR. This study describes the development of a novel RT-LAMP assay for the detection of AHSV. The AHSV RT-LAMP assay has an analytical sensitivity of 96.1% when considering an rRT-PCR cut-off value of CT  > 36, or 91.3% when no rRT-PCR cut-off is applied. Diagnostic sensitivity and specificity were 100%. This assay provides for a rapid and low cost AHS diagnostic for use in the field.

Antibody response in horses following experimental infection with West Nile Virus lineages 1 and 2.

West Nile virus (WNV) has re-emerged as an important pathogen for humans and horses, which are considered to be incidental 'dead-end' hosts. We have demonstrated that horses are susceptible to experimental infection with WNV and that horses infected with either WNV lineage 1 or lineage 2 elicit a similar antibody profile in serum samples. These data suggest that virus-neutralizing antibody responses persist for longer than WNV-specific IgM levels in serum and that there are not any notable differences in the antibody profile following experimental infection of horses with either WNV lineage 1 and lineage 2 viruses. Furthermore, the duration of IgM appears to be short-lived in horses and may be useful for identifying and differentiating recent infections from previously exposed animals.

Beneficial effects of synthetic KL4 surfactant in experimental lung transplantation.

The aim of this study was to investigate whether intratracheal administration of a new synthetic surfactant that includes the cationic, hydrophobic 21-residue peptide KLLLLKLLLLKLLLLKLLLLK (KL4), might be effective in reducing ischaemia-reperfusion injury after lung transplantation. Single left lung transplantation was performed in Landrace pigs 22 h post-harvest. KL4 surfactant at a dose of 25 mg total phospholipid·kg body weight⁻¹ (2.5 mL·kg body weight⁻¹) was instilled at 37°C to the donor left lung (n = 8) prior to explantation. Saline (2.5 mL·kg body weight⁻¹; 37°C) was instilled into the donor left lung of the untreated group (n = 6). Lung function in recipients was measured during 2 h of reperfusion. Recipient left lung bronchoalveolar lavage (BAL) provided native cytometric, inflammatory marker and surfactant data. KL(4) surfactant treatment recovered oxygen levels in the recipient blood (mean ± sd arterial oxygen tension/inspiratory oxygen fraction 424 ± 60 versus 263 ± 101 mmHg in untreated group; p=0.01) and normalised alveolar-arterial oxygen tension difference. Surfactant biophysical function was also recovered in KL4 surfactant-treated lungs. This was associated with decreased C-reactive protein levels in BAL, and recovery of surfactant protein A content, normalised protein/phospholipid ratios, and lower levels of both lipid peroxides and protein carbonyls in large surfactant aggregates. These findings suggest an important protective role for KL4 surfactant treatment in lung transplantation.

Expression and secretion of Bluetongue virus serotype 8 (BTV-8)VP2 outer capsid protein by mammalian cells.

VP2 is the outermost Bluetongue virus (BTV) antigenic protein, forming triskelion motifs on the virion surface. Although VP2 has been expressed successfully through many systems, its paracrine expression as a soluble form by mammalian cells represents a difficult task. In the present paper two fragments of VP2 have been expressed successfully into the medium of transiently transfected mammalian cells through a fusion peptides strategy. The crude conditioned medium containing the secreted peptide could be employed for immunodiagnostic assay development or vaccine purposes.

Aspectos jurídicos de los trasplantes en España / No disponible

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Los traspalantes del mito a la actualidad I / No disponible

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Los trasplantes del mito de la actualidad II / No disponible

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Hysteresis of a biomaterial: influence of sutures and biological adhesives.

We studied the changes in energy consumption of samples of calf pericardium, when joined or not joined by sutures and adhesives, by means of hysteretic cycles. Sixty-four samples were subsequently subjected to tensile stress until rupture. An overlapping suture sewn in the form of a rectangle presented an acceptable mean resistance to rupture of over 10 MPa, although lower than the mean values in an unsutured control series where the mean resistance surpassed 15 MPa. The contribution of an acrylic adhesive to the resistance to rupture was negligible. The sutured samples that were reinforced with adhesives and had not been subjected to hysteretic cycles prior to rupture showed an anisotropic behavior. This behavior appeared to be lost in all the samples that underwent hysteretic cycles. We found an inflection point in the stress/strain curve following the stepwise increase in the load, with a value greater than and proximate to the final load applied. This inflection should be analyzed by means of microscopy. Finally, the mathematical relationship between the energy consumed and the stress applied, the strain or deformation produced and the number of cycles of hysteresis to which the samples were subjected was established as the ultimate objective of this study. The bonding systems provoked a greater consumption of energy, with the greatest consumption corresponding to the first cycle in all the series assayed. An equation relating the energy consumption in a sample to the number of hysteretic cycles to which it was subjected was obtained. Its asymptote on the x-axis indicates the energy consumption for a theoretical number of cycles, making it possible to estimate the durability of the sample.

Durability of a cardiac valve leaflet made of calf pericardium: fatigue and energy consumption.

We studied the mechanical behavior of membranes of calf pericardium, similar to those employed in prosthetic valve leaflets, when subjected to tensile fatigue. The objective was to assess its durability, as a fundamental property of cardiac bioprosthesis, and analyze the energy consumption. For this purpose, the authors built a hydraulic simulator to subject a spherical valve leaflet made of calf pericardium to cyclic stress mimicking cardiac function. A total of 522 assays were performed in 40 samples, subjected to cyclic pressures greater than 6 atm, and 482 subjected to pressures ranging between 2 and 6 atm. The mathematical expression that establishes the relationship between the pressure exerted and the frequency was obtained. If we assume that the function is continuous, this equation provides the range of fatigue tolerated for a given number of cycles. Using the optimal values (the five highest values per series), the expression was found to be y = 9.95x(-0 1214) (R(2) = 0.955), where x represents the frequency in cycles per second and y the pressure in atmospheres. In addition, we established the mathematical relationship between the energy consumed and the frequency, which was a function of the pressure exerted, regardless of the region or zone from which the samples had been obtained. The methods of manual and morphology-based selection employed produced widely dispersed results. When a mechanical criterion was included, the similarity in the energy consumed during fatigue testing markedly improved the correlation, with a coefficient of determination between paired samples of R(2) = 0.7477. A mechanical criterion, such as energy consumption, can help to improve sample selection and produce more consistent results. Finally, we obtained the mathematical expression that relates the energy consumed to the pressure exerted and the number of cycles per second to which the valve leaflet was subjected. This procedure enables us to establish the limit to the energy that a biomaterial can consume over a period of time during which it is subjected to a working pressure and, thus, calculate more precisely its durability.

Resistance and stability of a new method for bonding biological materials using sutures and biological adhesives.

The valve leaflets of cardiac bioprostheses are secured and shaped by sutures which, given their high degree of resistance and poor elasticity, have been implicated in the generation of stresses within the leaflets, contributing to the failure of the bioprostheses. Bioadhesives are bonding materials that have begun to be utilized in surgery, although there is a lack of experience in their use with inert tissues or bioprostheses. Tensile testing is performed until rupture in samples of calf pericardium, a biomaterial employed in the manufacture of bioprosthetic heart valve leaflets. One hundred and thirty-two trials are carried out in three types of samples: intact or control tissue (n = 12); samples transected and glued in an overlapping manner with a cyanoacrylate (n = 60); and samples transected, sewn with a commercially available suture material and reinforced at the suture holes with the same cyanoacrylate (n = 60). Seven days after their preparation, 12 samples from each group, including the controls, are subjected to tensile testing until rupture and the findings are compared. In the stability study, groups of 12 each of the remaining 48 glued and 48 sutured and glued samples underwent tensile testing until rupture on days 30, 60, 90, and 120, after their preparation. The results show that bonding with the adhesive provided a resistance ranging between 1.04 and 1.87 kg, probably insufficient for use in valve leaflets, but also afforded a high degree of elasticity. After 120 days, both the glued and the sutured and glued series show excellent elastic behavior, with no rigidity or hardening of the pericardium. These samples present reversible elongation, or strain, when they surpass their elastic limit at rupture. This finding may be due to a load concentration that is damaging to the pericardium, to the behavior of the tissue as an amorphous material, or perhaps to both circumstances. These results need to be confirmed in future studies as they may be of value in the design and manufacture of cardiac bioprostheses.

Comparative study of the mechanical behaviour of a cyanoacrylate and a bioadhesive.

We compared the mechanical resistance of 18 samples of calf pericardium bonded with a 100 mm2 overlap, by two types of glues: a cyanoacrylate (Loctite 4011) and a bioadhesive (BioGlue). Comparative tensile testing was also carried out in 40 paired samples, 20 bonded with the cyanoacrylate and 20 unbonded controls. The findings at rupture showed a greater resistance of the calf pericardium glued with cyanoacrylate, with a mean tensile strength of 0.15 MPa vs. 0.04 MPa for the biological glue (p= 0.000). They also demonstrated a loss of resistance of the samples bonded with cyanoacrylate when compared with that of the unbonded other halves of the pairs: 0.20 MPa and 0.27 MPa vs. 19.47 MPa and 24.44 MPa (p < 0.001). The method of selection by means of paired samples made it possible to establish the equations that relate the stress and strain, or deformation, with excellent coefficients of determination (R2). These equations demonstrate the marked elastic behaviour of the bonded samples. Moreover, these findings show the cyanoacrylate to be superior to the biological glue, leading to the examination of the compatibility, inalterability over time and mechanical behaviour of the cyanoacrylate in sutured samples, as well as the study of the anisotropy of the biomaterial when bonded with a bioadhesive.

Resistance to tensile stress of a bioadhesive utilized for medical purposes: Loctite 4011.

Sutures are the materials presently employed to secure and give shape to the valve leaflets of cardiac bioprostheses. Their high resistance and low degree of elasticity in comparison with the calf pericardium of which the leaflets are made generates internal stresses that contribute to the failure of the bioprostheses. Biological adhesives are bonding materials that have begun to be utilized in surgery, although there is a lack of experience in their use with inert tissues or bioprostheses. We report our study of Loctite 4011, a biological glue composed of a cyanoacrylate that has been employed for medical purposes, in which samples of pericardium bonded with this adhesive were subjected to uniaxial tensile stress. The samples were glued in such a way as to leave an overlap of 1 cm2 between the surfaces of the tissue. The series included 83 samples: 12 tested 24 h after bonding, 17 after 45 days, 17 after 90 days, 19 after 106 days and 18 after 152 days. The samples subjected to deferred trials were preserved using three types of chemical substances: glutaraldehyde, glycerol or saline plus antibiotics. The mean resistance to rupture of the series tested 24 h after gluing was 0.15 MPa (1.47 machine kg). This resistance remained nearly unchanged, regardless of the preservation solution employed, for at least 152 days, the time at which the study ended. The stress-strain curves demonstrated a high degree of elasticity throughout the 152 days, a finding that was not influenced by the preservation solution. This adhesive showed a considerable resistance to tensile stress, although probably insufficient to replace sutures. However, it maintained a surprisingly high degree of elasticity in the samples. Perhaps the time has come to combine these two elements, sutures and adhesives, to improve the elasticity of the structure without a loss of resistance, and increase the durability of bioprostheses.

Experimental heterotopic heart transplantation: an easier technique.

AIM: The use of heterotopic heart transplantation (HHT) in experimental surgery is an extended method to evaluate cardiac graft viability. To study endothelial injury after ischemia-reperfusion, the technique described by Matsui et al. in 1988 for HHT was chosen. A modification, which consists of replacing the atrium-to-atrium anastomosis for the tip of a 30-Fr venous cannula, was developed with the purpose of using this technique in small pigs. Both techniques were compared. METHODS: Twenty-seven consecutive HHTs in 17- to 20-kg pigs using Matsui's original technique and the modified technique were performed. Ischemia time, bleeding volume, mean gradient and anastomosis complications were measured to compare both techniques. RESULTS: Statistically significant decreases in ischemia time, bleeding volume and mean gradient with the modified technique were found. Furthermore, there were two cases of suture dehiscence with the original technique. CONCLUSIONS: The replacement of the atrium-to-atrium anastomosis for the venous cannula is a modification that statistically decreases the ischemia time, blood loss and avoids suture complications. This modification makes Matsui's technique easier, faster and safer in small pigs and it may be used in bigger animals and in any kind of non-permanent anastomosis.

Detection of equine arteritis virus (EAV)-specific cytotoxic CD8+ T lymphocyte precursors from EAV-infected ponies.

Equine arteritis virus (EAV) causes a systemic infection in equids with variable outcome, ranging from subclinical infections to severe disease, and also has the capacity to induce abortion in pregnant mares and persistent infections in stallions. The serum virus-neutralizing antibody response that invariably develops in the infected animal lasts for many months or years and is believed to play an important role in virus clearance. However, very little is known about cellular immunity against EAV because of a lack of methods for evaluating these immune responses. In the present study, we describe methods for detecting cytotoxic T lymphocyte (CTL) precursors in the peripheral blood of EAV-convalescent ponies using a (51)Cr release cytolysis assay. Primary equine dermal cells, used as CTL targets, were shown to express MHC I but not MHC II and to retain (51)Cr efficiently and support EAV replication. Peripheral blood mononuclear cells (PBMC) collected from EAV-convalescent ponies that had been incubated with or without live EAV were used as effectors. EAV-induced PBMC cultures showed evidence of expansion and activation of lymphoblasts, with an increase in the CD8(+)/CD4(+) ratio in comparison with mock-induced PBMC. The cytotoxicity induced by EAV-stimulated PBMC was virus specific, showed genetic restriction, was mediated by CD8(+) T lymphocytes and could be detected for periods of 4 months to more than 1 year post-infection. These findings and methods will hopefully contribute to an understanding of virus-host interactions in horses, in particular the mechanisms of virus clearance occurring during EAV infection.

Comparison of the mechanical behaviors of biological tissues subjected to uniaxial tensile testing: pig, calf and ostrich pericardium sutured with Gore-Tex.

The purpose of this study was to compare the mechanical behavior of calf pericardium, pig pericardium and ostrich pericardium when subjected to tensile testing. Tensile stress was applied to 108 tissue samples, 36 of each type of tissue, until rupture. Groups of three adjacent strips measuring 12 x 2 cm(2) were cut longitudinally. Each group consisted of an unsutured center sample, or control, and the two contiguous samples, that on the right sutured with Gore-Tex at a 90 degrees angle with respect to the longitudinal axis and that on the left sewn with the same suture material at 45 degrees angle. The sutured samples showed a statistically significant loss of resistance (p<0.001) when compared with the corresponding unsutured tissue. The mean stresses at rupture for sutured ostrich pericardium were 21.81 and 20.81 MPa in the samples sewn at 45 degrees and 90 degrees, respectively, higher than those corresponding to unsutured calf and pig pericardium, 14.0 and 11.49 MPa, respectively, at rupture. The analysis of the stress/strain curve shows a smaller difference between sutured and unsutured ostrich pericardium than those observed in the other two biomaterials. These results demonstrate that, in addition to its greater resistance, ostrich pericardium also presents a less pronounced interaction with the suture material. Its capacity to absorb the shearing stress produced by the suture is greater. This report also confirms that the method of selection using paired samples ensures their homogeneity and makes it possible to predict the behavior of a sample by determining that of the other half of the pair.

Sutured calf pericardium: influence of the type and angle of the suture on mechanical behavior.

Careful selection of the biological tissue to be used in cardiac bioprostheses and a thorough knowledge of its mechanical behavior, facilitating both the prediction of this behavior and the interactions between the tissue and the other materials employed, is the best approach to designing a durable implant. For this purpose, a study involving uniaxial tensile testing of calf pericardium was carried out. Two sets of three contiguous strips of tissue were obtained from each pericardial membrane, to perform a total of 144 trials. Two samples were sewn with one of four commercially available suture materials: Gore-Tex, nylon, Prolene and silk. In each set of three samples, the center strip remained intact and unsutured to serve as a control, while the left-hand strip was sutured at a 45 degrees angle with respect to the longitudinal axis and the right-hand strip was sewn at a 90 degrees angle. All the samples were tested until rupture. The results demonstrated a significant loss of mean load (p<0.01) in the sutured samples at rupture. The angle of the suture had no influence on these results, although the stress/strain curves showed that, as the tensile stress increased, the mechanical behaviors were less uniform. The rupture of the collagen fibers could explain this phenomenon. The pericardium sutured with Gore-Tex presented a greater strain, or deformation (elongation), at lower levels of stress, regardless of the angle of the suture. The tissue selection criteria, based on the use of paired samples, enabled a correct prediction of the mechanical behavior of the tissue, with excellent correlation coefficients (>0.98) and a high degree of homogeneity in the results.

Mechanical behavior of chemically treated ostrich pericardium subjected to uniaxial tensile testing: influence of the suture.

The mechanical behavior of sutured ostrich pericardium was studied by uniaxial tensile testing. One hundred forty-four tissue specimens were assessed: 96 sutured samples (48 in which a centrally located suture was placed at an angle of 90 degrees with respect to the longitudinal axis, whereas in the remaining 48, a centrally located suture was placed at a 45 degrees angle to the longitudinal axis, in sets of 12 samples each, sewn with sutures made of Gore-Tex, nylon, Prolene, or silk), and 48 unsutured controls. Each group of 24 samples sewn at one angle or the other with the different suture materials was assayed together with a corresponding control group of 12 unsutured samples. The mean tensile strengths in the unsutured controls ranged between 30.16 MPa and 43.42 MPa, whereas those of the sutured sets ranged from 14.68 MPa to 21.91 MPa. The latter presented a statistically significant loss of resistance (p < 0.01) when compared with the unsutured tissue samples. The angle of the suture with respect to the longitudinal axis influenced the degree of shear stress produced by the suture, as well as the behavior of the different suture materials used. The set of samples sewn with Prolene appeared to be that most sensitive to changes in the angle of the suture, whereas tissue sewn at a 45 degrees angle with Gore-Tex presented lower shear stress values in comparison with samples in which the other three materials were used. A method of tissue selection based on morphological and mechanical criteria was used to ensure the homogeneity of the results in such a way that the coefficients of determination (R2) for the stress/strain curve fitting equation ranged between 0.888 and 0.995. This excellent fit made it possible, applying regression analysis, to predict the mechanical behavior of a specimen by determining that of a contiguous tissue sample. Thus, it should be possible, at least theoretically, to characterize the behavior of a specific region or zone of the biomaterial. In conclusion, ostrich pericardium exhibits strong resistance to rupture, even when sutured. The selection method used ensures the homogeneity of the samples and, thus, of the results. The angle of the suture with respect to the longitudinal axis, where the load is centered, determines the shear stress produced by the suture and the mechanical behavior of each suture material.