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Frontal sinuses are of special interest for personal identification thanks to their high variability. A common procedure for comparing ante-mortem and post-mortem material is based on coding systems classifying frontal sinuses according to their morphological and metrical characteristics. However, the calculation of possible combinations is performed on the hypothesis that all the classified features are independent one from each other. This study aims at analysing the correlation among morphological and metrical characteristics of frontal sinuses. Two hundred CT scans of patients equally divided between males and females were segmented through the ITK-SNAP software. Number of accessory septations, scalloping and supra-orbital cells, side asymmetry and superiority, breadth, height, length and volume were extracted from the frontal sinuses of each subject. A possible relationship among morphological and metrical features was analysed through Pearson's correlation test, Mann-Whitney test or chi-square test according to the type of compared data (p < 0.05). In general, a positive correlation was found for all comparisons among metrical measurements (breadth, height, depth and volume) and number of septations, scalloping and supra-orbital cells (p < 0.05), but not between the number of scalloping and supra-orbital cells. In addition, side of asymmetry was positively related with side of superiority of the upper profile of frontal sinuses (p < 0.05). This study proved that morphological and metrical characteristics of frontal sinuses are strictly related one with each other: therefore, the coding systems based on these features should be applied with caution for personal identification, as the number of possible combinations is lower than reported by literature.

Sphenoid sinus accessory septations involve serious complications during their removal for the injury of the internal carotid artery (ICA) and the optic nerve (ON). The relationships of this anatomical variant with sphenoid size still remain unclear. In the present study, 260 patients (equally divided among sexes, aged between 18 and 92 years) were retrospectively assessed. Number of accessory septations and their relationship with ICA and ON were recorded. The 3D model of sphenoid sinuses was extracted. Pearson's coefficient was calculated to assess correlations between the volume of sphenoid sinuses and the number of septations (P < 0.05). Sex-related differences in prevalence of septations inserted onto ICA or ON were assessed through chi-square test (P < 0.05). Differences in volume between patients with and without ICA or ON septal insertions were assessed through Mann-Whitney test (P < 0.05). In most of the cases, two septations were found (24.6%), whereas only 21.9% of patients did not show any septum. ICA and ON septal insertions were found in 20.8% and 7.7% of cases, respectively. Number of septations significantly increased with sinus volume, independently from sex (P < 0.001). Moreover, volume was significantly higher in patients with ICA septal insertion (P < 0.001), whereas no difference was found between subjects with and without ON septal insertion (P > 0.05). The present study first proved that septations and probability of ICA insertion are related with sphenoid volume. On the other side, ON insertion does not depend upon sphenoid sinuses size. Anat Rec, 303:1300-1304, 2020. © 2019 American Association for Anatomy., (© 2019 American Association for Anatomy.)

Purpose: Vidian neurectomy is a surgical procedure applied to different pathological conditions, including chronic rhinitis and sphenopalatine neuralgia. The choice of the correct surgical approach depends upon the possible protrusion of Vidian nerve into the sphenoid sinuses. The present study analyzes the possible relationship between protrusion of Vidian nerve and volume of sphenoid sinuses., Methods: In total, 320 maxillofacial CT-scans were retrospectively assessed. Subjects equally divided among males and females (age range 18-94 years) were divided into three groups according to the profile of Vidian nerve protrusion: type 1: Vidian nerve inside the sphenoid corpus; type 2: partially protruding into the sphenoid sinus; and type 3: entirely protruding into the sphenoid sinus through a stalk. Volume of sphenoid sinuses was extracted through the ITK-SNAP-free software and automatically calculated. Possible statistically significant differences in prevalence of the three types between males and females were assessed through Chi-squared test (p < 0.05). Differences in volume of sphenoid sinuses in subjects included within the three types were assessed through one-way ANOVA test (p < 0.05), separately for males and females., Results: Type 2 was the most prevalent (46.5%), followed by type 1 (38.8%) and type 3 (14.7%), without significant differences according to sex (p > 0.05). Volume significantly increased passing from type 1 to type 3 both in males (p < 0.01) and in females (p < 0.01)., Conclusions: The results prove the existence of a strict relationship between sphenoid sinuses pneumatization and protrusion of the Vidian canal and give a contribution to the knowledge of this important anatomical variant in endoscopic surgery.

Pharyngeal volume is important in anesthesiology for correctly assessing intubation procedures. However, most of studies are based on patients in upright position and do not assess possible relationships between pharyngeal volume and cranial size. This study aims at measuring pharyngeal volume in CT-scans and to assess possible statistically significant differences according to sex.Eighty healthy subjects (40 males and 40 females) aged between 21 and 86 years were retrospectively chosen from a hospital database of maxillofacial CT-scans; 3D segmentation was performed separately for naso-, oro- and laryngopharyngeal portion through ITK-SNAP software, and their volume was calculated. Three cranial measurements were obtained: distance between anterior and posterior nasal spine, upper facial height (nasion-prosthion) and biorbital breadth (ectoconchion-ectoconchion distance).The effect of sex on volume for each pharyngeal portion was assessed through one-way ANCOVA test using each of the 3 cranial measurements as covariate (P < 0.05).On average, the volume of nasopharynx, oropharynx and laryngopharynx was 7.2 ± 2.7 cm, 7.5 ± 4.2 cm, 3.5 ± 2.2 cm respectively in males, and 6.4 ± 2.9 cm, 5.2 ± 2.1 cm, 3.0 ± 1.8 cm in females. Statistically significant differences according to sex were found only for oropharyngeal volume, independently from cranial measurements (P < 0.05).This study provides data concerning volume of pharyngeal air space in supine subjects: these reference standards can be useful for anaesthesiologic procedures.

Sphenoid sinuses are considered the most variable structures of human body: therefore, they may be used for personal identification, through the application of 3D segmentation procedures. This study aims at proposing a new protocol for personal identification based on 3D-3D superimposition of sphenoid sinuses segmented from head CT scans. Adult subjects (equally divided among males and females) who underwent two head CT scans were extracted from a hospital database. Sphenoid sinuses were segmented through ITK-SNAP software and the corresponding 3D models were automatically superimposed to obtain 40 matches (when they belonged to the same person) and 260 mismatches (when they were extracted from different individuals). The RMS (root mean square) point-to-point distance was then calculated for all the superimpositions: differences according to sex and group (matches and mismatches) were assessed through the Mann-Whitney test (p < 0.05). On average, the RMS value was almost ten times smaller in matches (0.22 ± 0.11 mm) than in mismatches (2.16 ± 0.57 mm) with a statistically significant difference according to group (p < 0.05), but not to sex (p > 0.05). The study proposed a new method for assessing personal identification from segmented 3D models of sphenoid sinuses, useful in the forensic contexts where other methods might not be implementable or successful.

Anatomical uniqueness plays a significant role in the personal identification process of unknown deceased. Frontal sinuses have been widely used in the past decades for this purpose, mostly using 2D X-ray techniques. However, the modern 3D CT-based segmentation methods may help in developing novel and more reliable methods of identification. This study aims at assessing the anatomical uniqueness of frontal sinuses through the 3D model registration. Thirty subjects who underwent two maxillofacial CT scans (interval: 1 month to 5 years) were selected from a hospital database. Frontal sinuses were automatically segmented through ITK-SNAP open source software and the 3D models belonging to the same patient were automatically superimposed according to the least point-to-point difference between the two surfaces. Two hundred patients were randomly selected from the same database and undergo the same procedure to perform 200 superimpositions of frontal sinuses belonging to different individuals, equally divided between males and females (mismatches). Statistically significant differences of average root mean square (RMS) point-to-point distance between the group of matches and mismatches, as well as possible differences according to sex, were assessed through Mann-Whitney U test (p < 0.05). In the group of matches, RMS ranged between 0.07 and 0.96 mm (mean RMS 0.35 ± 0.23 mm), while in the group of mismatches, it ranged between 0.96 and 10.29 mm (mean RMS 2.59 ± 1.79 mm), with a statistically significant difference (p < 0.0001). Neither the matches nor the mismatches group showed statistically significant differences according to sex. This study proposes a novel 3D approach for the assessment of anatomical uniqueness of frontal sinuses, providing both morphological and quantitative analysis, and a new method of identification based on 3D assessment of frontal sinuses, applicable when ante-mortem CT scans are available.

Purpose: Anatomy of sphenoid sinuses has acquired a growing importance with the diffusion of transsphenoidal surgical procedures. A common risk in these practices is the damage of internal carotid artery (ICA) and optic nerve (ON), which may protrude into the sphenoid air cavities. This study aims at analysing the relationships between sphenoid sinuses volume and protrusion of ICA and ON., Methods: 260 head CT-scans were retrospectively analysed (equally divided among males and females, age range 20-92 years). Volume was segmented through ITK-SNAP software. In addition, the subjects were classified into four groups: no protrusion of any structure (group 1), protrusion of ICA (group 2), protrusion of ON (group 3), protrusion of both ICA and ON (group 4). Possible statistically significant differences in prevalence of the four groups according to gender were assessed through Chi-squared test (p < 0.05). Differences in volume between the four groups were assessed through one-way ANOVA test (p < 0.05), separately for males and females., Results: Group 1 was the most frequent (40.0%), followed by group 4 (27.7%) and group 2 (18.5%), without any difference according to gender. For what concerns volume, cases of ICA and concomitant ICA + ON protrusion had significantly larger sinuses, whereas isolated ON protrusion did not modify sinus volume., Conclusions: Results show that protrusion of ICA is positively related with the volume of sphenoid sinuses, whereas the same relation was not verified for ON: surgeons should accurately consider possible ON protrusion in each case, as it may occur independently from sphenoid sinuses volume.

Purpose: The pterygopalatine fossa is an important anatomical structure for several surgical and anaesthesiologic procedures; yet, very few data are available about its size. This study aims at providing a metrical assessment of pterygopalatine fossa through an innovative 3D segmentation procedure on head CT-scans., Methods: CT-scans from 100 patients (50 males and 50 females) aged between 18 and 85 years were chosen for the study. Right and left pterygopalatine fossae were segmented through ITK-SNAP open source software. Height and volume were calculated on the acquired 3D models. In addition, anterior-posterior nasal spine distance, upper facial height (nasion-prosthion) and biorbital breadth (ectoconchion-ectoconchion) were measured as well. Statistically significant differences of height and volume according to sex and side were assessed through two-way ANOVA test: sexually dimorphic measurements were further assessed through one-way ANCOVA test using the three cranial measurements as covariates (p < 0.05)., Results: On average pterygopalatine fossa height was 24.1 ± 3.5 mm in males, and 22.8 ± 3.4 mm in females, whereas volume was 0.930 ± 0.181 cm 3 in males and 0.817 ± 0.157 cm 3 in females, with statistically significant differences according to sex (p < 0.05), but not to side (p > 0.05); interaction was negligible for both the measurements. ANCOVA test verified that sexual dimorphism of both measurements is independent from general cranial size (p < 0.05)., Conclusions: The present study highlighted the sexual dimorphism of pterygopalatine fossa: results may improve the knowledge of this anatomical structure difficult to explore, but crucial in several fields of clinics and surgery.

Background: The intrapetrous carotid artery (IPCA) is one of the most unexplored anatomic regions, and its 3-dimensional reconstruction in living subjects is still missing. This study aims to describe the IPCA on 3D models extracted from head computed tomography (CT) scans., Methods: The intrapetrous carotid artery was manually segmented on head CT scans of 100 healthy patients free from vascular and neurologic pathologies (50 men and 50 women; age range, 18-91 years). Angles of the posterior and anterior genu, diameter and length of the horizontal portion, and volume of the entire canal were calculated through 3D analysis software. Statistically significant differences according to sex and side were assessed through 2-way analysis of variance (P < 0.05). Correlation of each measurement with age was calculated as well., Results: On average, the angles of the posterior and anterior genu were 120.1° ± 10.4° and 118.0° ± 10.0° in men and 119.5° ± 9.2° and 117.6° ± 10.3° in women, respectively, without statistically significant differences according to sex or side (P > 0.05). The average length and diameter of the horizontal part were, respectively, 25.5 ± 2.9 and 5.8 ± 0.8 mm in men and 24.0 ± 2.3 and 5.3 ± 0.8 mm in women. The volume of the IPCA was 0.941 ± 0.215 cm 3 in men and 0.752 ± 0.159 cm 3 in women. The length and diameter of the horizontal portion and the volume of the IPCA showed statistically significant differences according to sex (P < 0.05). No correlation with age was found., Conclusions: This study provides data concerning not only linear and angular measurements, but also volumes of the IPCA, which are useful in planning surgical interventions of the cranial base., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Anatomical variants of the ethmoid bone have a special importance in several fields, especially in otolaryngology; a precise understanding of the complex anatomy and anatomic variations of the ethmoid bone is crucial for radiological diagnosis of paranasal pathology and for surgical work-up in order to avoid potential complications. The present study aims at helping general radiologists to become familiar with the most common forms of ethmoid anatomical variants and their clinical relevance.

Improvements in computed tomography and in functional endoscopic sinus surgery have recently increased interest toward paranasal sinus anatomy and anatomic variations that can be observed in patients affected by sinusitis. Isolated sphenoid sinusitis is a relatively rare pathology, often related to nonspecific symptoms, therefore making diagnosis difficult. The correlation between this type of sinusitis and anatomical variants remains unclear.The authors' aim was to retrospectively revise paranasal sinuses computed tomography scans of patients affected by sphenoid sinusitis, compared with a control group, analyzing the types of sphenoid sinus and the presence of aberrant pneumatization, and performing a segmentation of the sphenoid sinuses to calculate the volumes.Sphenoid sinuses of 60 patients affected by sinus opacification, compared with a control group, were segmented. Type of sinus (sellar, presellar, postsellar) and presence of aberrant pneumatization were assessed as well. Possible statistically significant differences in volumes according to sex and group were assessed through 2-way ANOVA test (P < 0.05). Post-hoc test was assessed through Student t test. χ test was applied in order to verify the statistically significance of differences in frequency of different types of sinus pneumatization variants (P < 0.05).Average volume of sphenoid sinuses in males was of 7.672 cm and of 7.751 cm in females within the group of patients; statistically significant differences in volume were found according to sex (P: 0.342), but not between the patients and control group (P: 0.0929). Post-hoc test verified that males affected by sinus opacification showed smaller volumes in comparison with the control males (P < 0.05). In addition, patients by affected sinus opacification showed more frequently the postsellar type and were less affected by pneumatization variants of the sphenoid bone than the control group (P < 0.05).This study first suggests the possible protective role of variants of pneumatization in the development of sphenoid sinus opacification.

Aim Sella turcica bridging and ossified carotico-clinoid ligament are two variants of the sella turcica, the origin of which is partially unknown. These variations should be properly recognised, as they may hamper the removal of the anterior clinoid process in surgical procedures. Therefore, our aim was to determine the prevalence of these two anatomical variants and to investigate their prevalence according to patient sex and age in a series of maxilla computed tomography scans. Materials and methods We revised 300 computed tomography scans of the head from northern Italian patients, stratified into three age groups (18-40 years, 41-60 years, >60 years): a logistic regression analysis was used to explore an association of sella turcica bridging with age and sex through Matlab software, also including a test for the extracted model ( P < 0.05). Results The mean prevalence of sella turcica bridging and ossified carotico-clinoid ligament were 0.16 ± 0.06 (48/300, 16.0%) and 0.09 ± 0.03 (26/300, 8.7%), respectively. Statistically significant differences according to sex were found neither for sella turcica bridging ( P = 0.345) nor for ossified carotico-clinoid ligament ( P = 0.412). Only sella turcica bridging showed a correlation with age ( P = 0.007). In addition, the two variants were often associated, as patients without sella turcica bridging usually did not show ossified carotico-clinoid ligament ( P < 0.001). Discussion Our results suggest an association between the two variants, and provide a novel contribution to the debate around their origin.

Purpose: Surgical reconstruction of zygomatic bones in cases of traumatic injuries is a frequent event: assessment of symmetry is mandatory for a correct restoration of zygomatic shape, but the literature is discordant about its quantification. The purpose of this study is to show a novel method for assessing symmetry of zygomatic bone through mirroring of 3D models segmented on CT-scan., Materials and Methods: A total of 100 patients (50 male and 50 female), divided into two age groups (18-49 years and 50-92 years) were selected from the computed tomography (CT) scan database of a hospital in northern Italy. Zygomatic bones from each patient were segmented, and the left bone was automatically mirrored and registered on the right one according to the least point-to-point distance between the two surfaces. The mean and root mean square (RMS) distance between the two models was then calculated. Statistically significant differences according to sex and age groups were assessed through two-way analysis of variance (p < 0.05). In addition, the effect size of differences was calculated., Results: The method proved to be repeatable, with inter- and intraoperator errors lower than 5%. Overall, mean and RMS point-to-point distances were respectively 0.01 mm and 0.84 mm, without statistically significant differences according to sex or age (p > 0.05), and with negligible effect size., Conclusion: This study provides an innovative method for assessing the symmetry of the zygomatic bone based on surface analysis. Results may provide useful indications for the reconstruction of zygomatic bones in maxillofacial surgery., (Copyright © 2018 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.)

Purpose: Computed tomography represents the gold standard for the assessment of morphological characteristics of sphenoid sinuses, whose anatomy has acquired a novel interest because of the recent introduction of transsphenoidal surgery and robot-assisted procedures. One of the most relevant parameters for planning surgical intervention is the volume of sphenoid sinuses, and with time few population studies have been published. However, at present, no data are available concerning the relation between volume and anatomical variants of sphenoid sinuses., Methods: We retrospectively evaluated head CT-scans of 100 patients (age range 25-99 years; mean age males 45.0; mean age females 50.5 years) to calculate the volume of sphenoid sinuses through automatic segmentation. Possible statistically significant differences according to sex and variants of pneumatization, and type of sinus were assessed, respectively, through Student's t test and one-way ANOVA test (p < 0.05)., Results: Average volume of sphenoid sinuses in males was 10.005 ± 5.101 cm 3 , in females 7.920 ± 3.176 cm 3 . Differences according to sex were statistically significant (p < 0.05). Patients with pneumatization of pterygoid processes, dorsum sellae and anterior clinoid processes had a significantly higher volume than unaffected subjects. Moreover, differences of volume according to the type of sphenoid sinus were statistically significant (p < 0.05)., Conclusions: Results show that volume of sphenoid sinuses strongly depend upon the type of sinus and possible pneumatization variants. Moreover, the important of ethnic variability is confirmed.

Sphenoid bone may be affected by different variants of pneumatisation, which have a relevant importance from a clinical and surgical point of view. The description of such variants in different populations may give useful information. However, few articles describe the variability of sphenoid pneumatised structures and none of them focuses on Northern Italian population. Variants of pneumatisation of sphenoid bone were described in a sample of 300 Northern Italian patients who underwent a CT scan. More than fifty-seven percent of patients showed a form of anatomical variant: the most common form was the pneumatised pterygoid processes (39.6%), followed by dorsum sellae (32.9%) and clinoid processes (20.3%), without statistically significant differences between males and females (p > 0.01). In 26.3% of patients, a combined pneumatisation of these three structures was observed, being the combination pterygoid processes-dorsum sellae the most frequent (11.3%). In 9.3%, all the three sphenoid structures were affected. This article is the first description of the prevalence of different variants of pneumatisation in a Northern Italian population: the occurrence of such forms has to be acknowledged for their possible clinical and surgical consequences.

Babesia ovis, a tick-transmitted intraerythrocytic protozoan parasite, causes severe infections in small ruminants from Southern Europe, Middle East, and Northern Africa. With the aim of finding potential targets for the development of control methods against this parasite, sequence analysis of its genome led to the identification of four putative cysteine proteases of the C1A family. Orthology between B. ovis, B. bovis, T. annulata, and T. parva sequences showed that each B. ovis C1A peptidase sequence clustered within one of the four ortholog groups previously reported for these piroplasmids. The ortholog of bovipain-2 of B. bovis and falcipain-2 of Plasmodium falciparum, respectively, was designated "ovipain-2" and further characterized. In silico analysis showed that ovipain-2 has the typical topology of papain-like cysteine peptidases and a highly similar predicted three dimensional structure to bovipain-2 and falcipain-2, suggesting susceptibility to similar inhibitors. Immunoblotting using antibodies raised against a recombinant form of ovipain-2 (r-ovipain-2) demonstrated expression of ovipain-2 in in vitro cultured B. ovis merozoites. By immunofluorescence, these antibodies reacted with merozoites and stained the cytoplasm of infected erythrocytes. This suggests that ovipain-2 is secreted by the parasite and could be involved in intra- and extracellular digestion of hemoglobin and/or cleavage of erythrocyte proteins facilitating parasite egress. A significant reduction in the percentage of parasitized erythrocytes was obtained upon incubation of B. ovis in vitro cultures with anti-r-ovipain-2 antibodies, indicating an important functional role for ovipain-2 in the intra erythrocytic development cycle of this parasite. Finally, studies of the reactivity of sera from B. ovis-positive and negative sheep against r-ovipain-2 showed that this protease is expressed in vivo, and can be recognized by host antibodies. The results of this study suggest that ovipain-2 constitutes a potential target for immunotherapies and drug development against ovine babesiosis., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Intercountry adoptions from Guatemala were highly controversial, because of the large numbers of children being adopted to the USA, along with evidence of corruption and child theft. Since the implementation of the Hague Convention on Intercountry Adoption in 2008, Guatemala's central authority for adoption has prioritized domestic placements for children over intercountry adoption. A possible attitudinal barrier to domestic adoption in Guatemala-negative attitudes and prejudice against Indigenous people-was investigated through questionnaires measuring attitudes toward adoption and attitudes toward and social distance from the two major ethnic groups (Ladino and Indigenous). Guatemalan university students (N = 177, 61 % men) were recruited from basic required courses at a private university. Results showed that attitudes toward adoption in general were more favorable than toward interethnic adoption, with the most negative attitudes toward adoption of Ladino children by Indigenous parents. Multiple regression and analysis of covariance models revealed that female gender, experience with adoption and more positive attitudes about Indigenous persons were associated with more positive attitudes toward adoption. The findings imply that negative attitudes toward Indigenous persons are associated with negative attitudes toward adoption, and serve as barriers to promoting domestic adoption in Guatemala.

Objective: To identify risk factors for death among patients with severe asthma., Methods: This was a nested case-control study. Among the patients with severe asthma treated between December of 2002 and December of 2010 at the Central Referral Outpatient Clinic of the Bahia State Asthma Control Program, in the city of Salvador, Brazil, we selected all those who died, as well as selecting other patients with severe asthma to be used as controls (at a ratio of 1:4). Data were collected from the medical charts of the patients, home visit reports, and death certificates., Results: We selected 58 cases of deaths and 232 control cases. Most of the deaths were attributed to respiratory causes and occurred within a health care facility. Advanced age, unemployment, rhinitis, symptoms of gastroesophageal reflux disease, long-standing asthma, and persistent airflow obstruction were common features in both groups. Multivariate analysis showed that male gender, FEV1 pre-bronchodilator < 60% of predicted, and the lack of control of asthma symptoms were significantly and independently associated with mortality in this sample of patients with severe asthma., Conclusions: In this cohort of outpatients with severe asthma, the deaths occurred predominantly due to respiratory causes and within a health care facility. Lack of asthma control and male gender were risk factors for mortality.

Anaplasma species are obligate intracellular rickettsial pathogens transmitted by ticks with an impact on human and animal health. Anaplasma ovis infects sheep and goats in many regions of the world, and it can be diagnosed by different methods like Giemsa staining, PCR or competitive ELISA. In this study, a PCR based on the gene coding for major surface protein 4 (MSP-4) was used to examine field samples collected from sheep in different countries. Altogether, 1161 blood samples from Turkey (n = 830), Iraq (n = 195), Sudan (n = 96) and Portugal (n = 40) were examined, of which 31.4%, 66.6% 41.6% and 82.5%, respectively, were positive. This indicates high prevalence of A. ovis in the countries under investigation, and it can be assumed that the situation in other areas of the world might be similar. Thus, A. ovis should be considered as an important constraint of livestock production, and further efforts are needed to better understand the epidemiology and to implement suitable control measures., (© 2013 Blackwell Verlag GmbH.)

Chondroitin-4-sulphate (ChS A) was immobilized by matrix assisted pulsed laser evaporation (MAPLE) with the aid of a UV KrF* excimer laser source. Distilled water was used as solvent for the preparation of the frozen composite MAPLE targets. The surface morphology, chemical structure and functional properties of laser transferred ChS A were investigated as a function of laser processing conditions. The results indicate that the amount of laser immobilized material, structure, and functional properties can be controlled by the laser fluence value used for the irradiation of the MAPLE targets. Under selected irradiation conditions besides the molecular structure, the functional properties of the laser processed ChS A molecules can be maintained., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bovine serum is an essential factor for the continuous in vitro growth of Babesia bovis parasites. Culture media typically contain 40% (v/v) of bovine serum. In the present study assays with low-serum media were performed. The growth of B. bovis Mo7 was successively lower in media with 30%, 20% and, principally, with 10% of serum. Without serum, the parasites were not able to propagate. In media with 10% of serum, the supplementation with Albumax II improved visibly the growth of B. bovis at the end of each cycle. Regarding the addition of orotic acid, no considerable effect was observed in media with 20% or 10% of serum, with or without Albumax II. B. bovis parasites cultured in vitro in all these media maintain their typical morphology during the intraerythrocytic stages.

Dielectrophoresis is a method that has demonstrated great potential in cell discrimination and isolation. In this study, the dielectrophoretic sorting of normal and Babesia bovis infected erythrocytes was performed using a microfabricated flow cytometer. Separation was possible through exploitation of the dielectric differences between normal and infected erythrocytes, essentially due to the higher ionic membrane permeability of B. bovis infected cells. Sorting experiments were performed inside a microchip made from Pt microelectrodes and SU-8 channels patterned on a glass substrate. Optimum cell separation was achieved at 4 MHz using an in vitro culture of B. bovis suspended in 63 mS/m phosphate buffer and applying a sinusoidal voltage of 15 V peak-to-peak. Normal erythrocytes experienced stronger positive dielectrophoresis (pDEP) than B. bovis infected cells, moving them closer to the microelectrodes. Under these conditions it was possible to enrich the fraction of infected cells from 7 to 50% without the need of extensive sample preparation or labelling. Throughout the experiments very few microliters of sample were used, suggesting that this system may be considered suitable for integration in a low-cost automated device to be used in the in situ diagnostic of babesiosis.

Quality and safe meat production and livestock husbandry are important foci for addressing the wider underlying economic and political challenges. In the last few years, an intense focus of the scientific community has been placed on breakouts of livestock diseases especially in Asia, which have spread into neighbouring countries including Europe. These outbreaks had a serious impact on the livelihood of the farmers as well as the economy of the affected countries. Given this, the establishment of a network of diagnostic facilities is a great demand both at the national and regional levels. In most of the cases, diagnostic assays are either not available or they are not validated. The aim of this collaborative network was to: 1 Distribute and harmonize diagnostic tools required for pathogen detection and differentiation. 2 Build the capacity to ensure the conduction of integrated disease control measures.

We present a particle-sorting device based on the opposition of dielectrophoretic forces. The forces are generated by an array of electrode chambers located in both sidewalls of a main flow channel. Particles with different dielectric response perceive different force magnitudes and are therefore continuously focused to different streamlines in the flow channel. We relate the particles' dielectric response to their output position in the downstream channel. We demonstrate the performance of the device by separating a mixed yeast cell population into pure fractions of viable and nonviable cells. Finally, we use the device to enrich red blood cells infected with Babesia bovis, a major pathogen in cattle and simultaneously confirm the hypothesis that infection with B. bovis causes significant changes in the dielectric response of red blood cells.

Impedance spectroscopy is a powerful tool for label-free analysis and characterisation of living cells. In this work, we achieved the detection of Babesia bovis infected red blood cells using impedance spectroscopy on a microfabricated flow cytometer. The cellular modifications caused by the intracellular parasite result in a shift in impedance which can be measured dielectrically. Thus, a rapid cell-by-cell detection with microliter amounts of reagents is possible. Unlike other diagnostic tests, this method does not depend on extensive sample pre-treatment or expensive chemicals and equipment.

Objective: To establish optimal conditions for long-term culture of the erythrocytic stage of Theileria uilenbergi., Sample Population: Red blood cells from 3 splenectomized sheep experimentally infected with a blood stabilate of T uilenbergi., Procedures: Cultures of T uilenbergi were initiated by use of blood from experimentally infected sheep collected when parasites were detected in Giemsa-stained thin blood smears. Different culture conditions were tested to optimize in vitro growth of the organisms. Subcultures were performed at a ratio of 1:2, 1:4, and 1:8 when the percentage of parasitized erythrocytes (PPE) was at least 1% or when the initial PPE was doubled., Results: The optimal culture medium was HL-1 medium (a complete chemically defined medium) supplemented with 20% sheep serum and 0.75% chemically defined lipids. Optimal culture conditions included incubation in a humidified 2% O(2), 5% CO(2), and 93% N(2) atmosphere at 37 degrees C. Cultures of the merozoite stage of the parasite were continuously propagated in vitro for > 1 year. The PPE reached values of up to 3%., Conclusions and Clinical Relevance: Optimization of culture conditions to reach a high PPE seems worthwhile. The continuous propagation of T uilenbergi in culture allows the production of parasite material without infecting animals and provides a continuous laboratory source of parasites for further studies.

Homologues to previously described Theileria (T.) annulata genes (T. annulata surface protein [TaSP], putative T. annulata membrane protein [TaD]) were successfully amplified by polymerase chain reaction (PCR) from Theileria sp. (China) merozoite cDNA, with 88% identity to TaD; TcSP partial cDNA, 94% identity to TaSP. Moreover, homologues to a secretory protein of T. annulata (TaSE), with a sequence identity of 99% on the cDNA level (TcSE partial cDNA) and to a potential membrane protein of T. lestoquardi (Clone-5), with a sequence identity of 100% on the genomic level (Tc Clone-5) but lacking an intron at positions 1894-1928 were identified.

A spherical porous glass support Trisoperl (TRISO) with four pore diameters (ø 47.8; 55.9; 102.6, and 108.8 nm) was characterized and selected for application in an optical flow cell immunosensor, in comparison with controlled pore glass (CPG). The TRISO support was functionalized with aldehyde and isothiocyanate (-NCS) groups to attach bovine serum albumin and alkaline phosphatase (AP). The TRISO isothiocyanate pore diameter 47.8 nm (TRISO(-NCS) 47.8 nm) showed the better potential to be used in the immunosensor. It immobilized more protein (19.3 mg AP per g support) while presenting an optical performance comparable to the CPG. CPG(-NCS) and TRISO(-NCS) 47.8 nm were tested in the immunosensor model where the saturation of the Goat IgG immobilized in the supports with Monoclonal Anti-Goat IgG conjugated with Cyanine-5 was reached, followed by regeneration with the elution buffer modified PBS pH 2.0. The TRISO(-NCS) 47.8 nm presented lower fluorescence intensity at saturation (around 39 AU) than CPG(-NCS) (150 to 104 AU), but revealed a major advantage related to the uniform arrangement of the spherical particles in the flow cell, generating no significant fluorescence differences between gravity and flow package.

The antigenic proteins of the piroplasm stage of Theileria species (China), the causative agent of theilerosis of small ruminants in China, were analyzed by Western blot, revealing several specific immunoreactive proteins of different predicted molecular weights. Furthermore, sera from Theileria species (China)-infected animals were probed for reactivity with the TaSP protein of T. annulata, for which a homologue has been described in Theileria species (China). Affinity chromatography demonstrated the presence of TaSP-reactive antibodies, and the majority of the sera showed reactivity with this protein both in Western blots and in ELISA. The identified parasite antigens and TaSP will be assessed for their suitability for developing diagnostic methods as well as evaluated for their capacity to stimulated host immune competent cells.

Immunosensors can play an important role in the improvement of veterinary diagnostics in areas such as the diagnosis of diseases, drug detection and food quality control, by providing applications with rapid detection, high sensitivity and specificity. Associated with advances in biochemistry, biotechnology, electronics and microfabrication, new transduction devices that translate a biological interaction into an electrical signal have been developed. An overview of the current immunoassay techniques used in standard diagnosis is presented. This includes a brief description of the different immunosensor transducer principles and some examples of present and future developments.

The increasing concentration of nitrite in groundwater, rivers and lakes brings serious risks to the public health and to the environment. The aim of this work was the development of an optical biosensor for quantifying nitrite based on the activity of cytochrome cd(1) nitrite reductase immobilised in controlled pore glass (CPG) beads. The developed biosensor operates by measuring the optical reflectance of nitrite reductase, which shows spectroscopic changes when nitrite reversibly binds to the reduced form and oxidizes the enzyme. The optimisation of the immobilisation procedure showed that the immobilisation efficiency is highly dependent on the pH, being very low at basic pH, and that the maximum capacity of the CPG for the immobilisation of cd(1) was estimated in 57+/-10 mg cd(1)/g CPG. The CPG/cd(1) specific activity remained stable at 4 degrees C, decreasing only 10% in 15 days. No observed effects of the immobilisation on the enzyme characteristics were detected, regarding both the red/ox absorbance spectra and the enzyme specific activity, since the red/ox spectra are in good agreement with similar ones obtained for cd(1) in solution, and the specific activity at time zero (0.6 micromoles of NO(2)(-) reduced min(-1) mg of protein(-1)) is similar to that found for the soluble enzyme. The biosensor shows a sensitive response to increasing concentrations of nitrite in solution, especially at 460 nm, at which it showed higher sensitivity. The corresponding detection limit of 0.93 microM is well below the maximum admissible concentration imposed by European Community norms, of 2.2 microM.

Nitrite is an important human health and environmental analyte. As such, the European Union (EU) has imposed a limit for nitrite in potable water of 0.1 mg l-1 (2.18 microM). In order to develop an optical biosensing system for the determination of nitrite ions in environmental waters, cytochrome cd1 nitrite reductase has been extracted and purified from the bacterium Paracoccus pantotrophus. The protein has been spectroscopically characterised in solution and important kinetic parameters of nitrite reduction of the cytochrome cd1 enzyme, i.e., Km, Vmax and kcat have been determined. The influence of pH on the activity of the cytochrome cd1 has been investigated and the results suggest that this enzyme can be used for the determination of nitrite in the pH range 6-9. Biosensing experiments with the cytochrome cd1 in solution suggested that the decrease in intensity of the absorption band associated with the d1 haem (which is the nitrite binding site), at 460 nm, with increasing nitrite concentrations would enable the measurement of this analyte with the optimum limit of detection. The cytochrome cd1 has been encapsulated in a bulk sol-gel monolith with no structural changes observed and retention of enzymatic activity. The detection of nitrite ions in the range 0.075-1.250 microM was achieved, with a limit of detection of 0.075 microM. In order to increase the speed of response, a sol-gel sandwich thin film structure was formulated with the cytochrome cd1. This structure enabled the determination of nitrite concentrations within ca. 5 min. The sol-gel sandwich entrapped cytochrome cd1 enzyme was found to be stable for several months when the films were stored at 4 degrees C.

An improved solid-phase enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies was developed to detect an African swine fever virus protein (VP73) in pig samples. The use of monoclonal antibodies against VP73 allowed a sensitive and specific sandwich ELISA. This assay detected a limiting antigen concentration of 0.05 microgram/ml of VP73, lower than the detection limit of 0.6 microgram/ml obtained by using polyclonal antibodies by the same ELISA. The whole virus particle was detected with this method to a limit of 2.3 x 10(2) PFU/ml. At the same time, an indirect ELISA was developed to detect ASFV antibodies. The results also indicate that this method may be a useful technique for epidemiological surveys.