Early induction of luteolysis in a timed AI protocol increases reproductive performance in beef cows.

The aim of this study was to produce a longer proestrus by early administration of prostaglandin F2α (PGF) in a timed artificial insemination (TAI) protocol in non-suckling Bos taurus (Angus crossbreed) beef cows. On day 0, cows (n = 489) were treated with an intravaginal 1 g progesterone (P4) device and 2 mg of estradiol benzoate. On day 7, cows were randomized into two groups: PGF7(n = 244; 500 µg of sodium cloprostenol 24 h before P4 device removal) or PFG8 (n = 245; 500 µg of sodium cloprostenol at P4 device removal). On day 8, P4 device was removed and cows received 0.5 mg of estradiol cypionate. All cows were submitted to TAI on day 10 (48-50 hours after P4 device removal). Cows treated with PGF on day 7 had greater expression of estrus (91.3 vs 79.1 %; P = 0.0011), regardless of CL presence at beginning of the protocol. Cows from PGF7 group had lower circulating P4 concentrations on day 8 in comparison with PGF8 treated cows (1.86 vs 2.99 ng/mL; P < 0.001). However, preovulatory follicle diameter did not differ among treatments at TAI (11.9 vs 11.8 mm; P = 0.7881). Pregnancy per TAI (P/TAI) was greater for PGF7 (63.9 vs 50.6 %; P = 0.0114) than PGF8 treated cows. In cows with follicles <8.5 mm at TAI, expression of estrus (33.3 vs 26.6 %; P = 0.6427) and P/TAI (40 vs 26.6 %; P = 0.3657) were low in both PGF7 and PGF8 treated cows, respectively. In cows with medium follicle size (8.5 to 11.9 mm) PGF7 treated cows had greater expression of estrus (90.5 vs 80 %; P = 0.033) and P/TAI (62.2 vs 49 %; P = 0.053). In cows with follicles >12 mm, expression of estrus was greater for PGF7 than PGF8 treated cows (99.1 vs 93.3 %; P = 0.045), however P/TAI did not differ (68.2 vs 59 %; P = 0.149). In cows with P4 < 1.99 ng/mL on day 8, expression of estrus was similar between PGF7 and PGF8 treated cows (92.6 vs 90.4 %; P = 0.53), and P/TAI tended to be greater for PGF7 than PGF8 treated cows (63 vs 52.1 % P = 0.076). However, in cows with P4 > 2 ng/mL PGF7 cows had higher expression of estrus (89 vs 67.5 %; P = 0.0005) and P/TAI (64.8 vs 48.7 %; P = 0.021) than PGF8. Thus, increasing the proestrous period by inducing luteolysis 24 hours earlier than removing the P4 intravaginal device enhanced fertility in non-suckling cyclic beef cows by increasing expression of estrus and P/TAI.

Comprehensive microRNA expression analysis of pediatric gonadal germ cell tumors: unveiling novel biomarkers and signatures.

microRNAs (miRNAs) are small endogenous noncoding RNAs, and alterations in their expression may contribute to oncogenesis. Discovering a unique miRNA pattern holds the potential for early detection and novel treatment possibilities in cancer. This study aimed to evaluate miRNA expression in pediatric patients with gonadal germ cell tumors (GCTs), focusing on characterizing the miRNA profiles of each histological subtype and identifying a distinct histological miRNA signature for a total of 42 samples of pediatric gonadal GCTs. The analysis revealed distinct miRNA expression profiles for all histological types, regardless of the primary site. We identified specific miRNA expression signatures for each histological type, including 34 miRNAs for dysgerminomas, 13 for embryonal carcinomas, 25 for yolk sac tumors, and one for immature teratoma, compared to healthy controls. Furthermore, we identified 26 miRNAs that were commonly expressed in malignant tumors, with six miRNAs (miR-302a-3p, miR-302b-3p, miR-371a-5p, miR-372-3p, miR-373-3p, and miR-367-3p) showing significant overexpression. Notably, miR-302b-3p exhibited a significant association with all the evaluated clinical features. Our findings suggest that miRNAs have the potential to aid in the diagnosis, prognosis, and management of patients with malignant GCTs.

Influence of taurine on the zootechnical performance and health parameters of juvenile Nile tilapia in a recirculating aquaculture system.

Taurine is considered a conditionally essential amino acid for fish, so its supplementation may improve feed conversion. This study evaluated the supplementation of taurine on growth performance, hematological and immunological parameters, production costs, and survival of Nile tilapia (Oreochromis niloticus) juveniles raised in a recirculating aquaculture system (RAS). A control diet was formulated with 360 g kg-1 of crude protein without fish meal and without taurine supplementation (Control). From the control diet, another diet supplemented with 9.7 g of taurine per kg of feed (Taurine) was produced. Fish fed diet supplemented with taurine had lower daily average weight gain and final average weight compared to the control diet (p < 0.05). It was observed that taurine had no influence on condition factor, survival, or hemato-immunological parameters of Nile tilapia juveniles, but there was a higher mean corpuscular volume and greater nitrogen retention in fish from the control group (p < 0.05). It is concluded that Nile tilapia juveniles do not benefit from taurine supplementation in RAS, even when fed diet containing plant-based protein sources.

Identifying malignant mesothelioma by a pathological survey using the São Paulo state hospital cancer registry, Brazil.

OBJECTIVE: To review the pathological diagnosis of possible cases and/or hidden cases of malignant mesothelioma (MM) between 2000 and 2012 using the Hospital-Based Cancer Registry database in the state of São Paulo, Brazil. METHODS: Possible cases were retrieved by assessing the database. Inclusion criteria were being older than 30 years of age and having ICD-O-3 topography and morphology codes related to MM. A board of expert pathologists reviewed the pathology reports and requested paraffin blocks in cases that demanded revision. After staining with calretinin, D2-40, WT-1 (as positive MM markers) and Ber-EP4 and MOC31 (as negative MM markers), cases were divided and studied independently by a pair of pathologists to confirm or discard the diagnosis of MM. RESULTS: Our sample comprised 482 cases from 25 hospitals, and 130 needed further histological revision. We received 73 paraffin blocks with adequate material. After board analysis, there were 9 cases with a definitive diagnosis of MM, improving the diagnostic rate in 12%. Two cases of previously diagnosed MM were discarded by review. CONCLUSIONS: Our results confirm that part of MM underdiagnosis and underreporting in Brazil is due to incomplete or mistaken pathological diagnosis.

Results of the Last 5 Years (2018-2022) of the Specialist Title Exam of The Brazilian College of Surgeons.

The article discusses the evolution of the Brazilian College of Surgeons (CBC) specialist title exam, highlighting the importance of evaluating not only theoretical knowledge, but also the practical skills and ethical behavior of candidates. The test was instituted in 1971, initially with only the written phase, and later included the oral practical test, starting with the 13th edition in 1988. In 2022, the assessment process was improved by including the use of simulated stations in the practical test, with the aim of assessing practical and communication skills, as well as clinical reasoning, in order to guarantee excellence in the assessment of surgeons training. The aim of this study is to demonstrate the performance of candidates in the last five years of the Specialist Title Test and to compare the performance results between the different surgical training groups of the candidates. The results obtained by candidates from the various categories enrolled in the test in the 2018 to 2022 editions were analyzed. There was a clear and statistically significant difference between doctors who had completed three years of residency recognized by the Ministry of Education in relation to the other categories of candidates for the Specialist Title..

Pedal Acceleration Time An Alternative Tool To Ankle-Brachial Index In Peripheral Arterial Disease.

INTRODUCTION: Ankle-Brachial Index (ABI) is a well-established diagnostic tool for evaluating peripheral arterial disease (PAD). Limitations in its application led to the development of alternative diagnostic methods, including Toe-Brachial Index (TBI) and Transcutaneous Pressure of Oxygen (TcPO2), yet these are not as widely available as ABI. Recently, Pedal Acceleration Time (PAT), has gained popularity as a new tool to assess PAD, requiring only an ultrasound. This study seeks to further establish the correlation between ABI and PAT, determining whether PAT can be a reliable alternative for diagnosing and assessing the severity of PAD. METHODS: ABI and PAT were measured in patients attending our consult with no history of vascular or endovascular surgery. Limbs with unmeasurable ABI were excluded. Patients were categorized into groups based on their PAD stage according to the Fontaine classification. Patient demographics, comorbidities and respective ABI and PAT were analysed. RESULTS: Sixty-nine patients (114 limbs) were included in the study. Mean age 68 ± 11.7 years, 78.3% male and 33.3% diabetic patients. Fifty-three claudicant limbs (46.5%) and 26 limbs (22.8%) with chronic limb threatening ischemia. Pearson correlation coefficient between ABI and PAT, showed a strong negative correlation (r= -0.78; p<0.01). Mean ABI and PAT for limbs in Fontaine stage I were 0.94 ± 0.17 and 82.0 ± 27.4 ms; Fontaine stage IIa 0.69 ± 0.21 and 141.3 ± 57.8 ms; Fontaine stage IIb 0.54 ± 0.14 and 173.4 ± 65.1 ms; Fontaine stage III 0.43 ± 0.15 and 216 ± 33.2 ms; Fontaine stage IV 0.49 ± 0.17 and 206.7 ± 78.1 ms, respectively. CONCLUSION: Our study suggests an inverse correlation between ABI and PAT, in accordance with the findings published in the literature, thus supporting the use of PAT as an easily reproducible and efficient alternative to ABI for evaluating the severity of PAD.

Combined Use of Fatty Acid Profiles and Elemental Fingerprints to Trace the Geographic Origin of Live Baits for Sports Fishing: The Solitary Tube Worm (Diopatra neapolitana, Annelida, Onuphidae) as a Case Study.

Diopatra neapolitana Delle Chiaje, 1841 (Annelida, Onuphidae) is one of the most exploited polychaete species in European waters, particularly in Ria de Aveiro, a coastal lagoon in mainland Portugal, where the overexploitation of this resource has led to a generalized decline of local populations. In an attempt to reduce the impact of harvesting, several management actions were implemented, but illegal poaching still fuels a parallel economy that threatens the sustainable use of this marine resource. The present study evaluated the combination of fatty acid profiles and elemental fingerprints of the whole body and jaws, respectively, of D. neapolitana collected from four harvesting locations within Ria de Aveiro in order to determine if their geographic origin could be correctly assigned post-harvesting. Results showed that both fatty acid profiles and elemental fingerprints differ significantly among locations, discriminating the geographic origin with higher accuracy when combining these two natural barcodes than when employing each individually. The present work can, therefore, contribute to the implementation of an effective management plan for the sustainable use of this marine resource, making it possible to detect if D. neapolitana was sourced from no-take zones and if it was collected from the place of origin claimed by live bait traders.

Factors associated with adherence to the principles of responsible companion animal guardianship in a municipality in southeastern Brazil.

Responsible companion animal guardianship (RCAG) covers aspects that are relevant to both animal and human health. Understanding the factors associated with adherence to RCAG principles can guide accountability, animal welfare and disease control. The present observational study describes the level of adherence to RCAG by guardians living in a medium-sized Brazilian municipality and identifies the factors associated with inadequate guardianship. Questionnaires were administered to randomly selected guardians of 704 dogs. The binary outcome of the study, namely more and less adequate compliers to RCAG principles, was analyzed using a score list comprising eight variables relating to the care provided to the dogs, namely provision of veterinary services, vaccination against rabies, deworming, no access to the streets without supervision, walking with guardian, dog freedom at home, registration (microchipping) and satisfactory food supply. Factors possibly associated with less adequate adherence to RCAG by the guardian, such as socioeconomic and cultural features of the guardians, characteristics of the dogs and attributes of the guardian-animal interactions, were analyzed using multiple logistic regression models. The RCAG actions least adopted by guardians were animal registration, provision of veterinary care, walking with the dog and preventing access of the dogs to the streets without supervision. Individuals who cared for a single dog, a mixed breed dog or had previously lost a dog were less likely to show adequate adherence to RCAG. Conversely, guardians who owned cars, acquired dogs as puppies, lived in households with a maximum of four residents, cared for a sterilized dog or thought that caring for a dog was easier than (or as) expected, had a higher chance of showing adequate adherence to RCAG. The results verify that the socioeconomic and behavioral characteristic of guardians must be taken into consideration for understanding the adherence to RCAG. In addition, it is important to facilitate access to veterinary services and to raise awareness about the significance of a safe and healthy environment for companion animals.

Chemical Composition and Acaricidal Activity of Essential Oil of Lavandula dentata L. on Engorged Females of Rhipicephalus (Boophilus) microplus (Acari: Ixodidae).

PURPOSE: Rhipicephalus (Boophilus) microplus is one the most significant ectoparasite in cattle farming in tropical and subtropical regions, causing problems to livestock health worldwide. The control of this ectoparasite primarily relies on the use of synthetic acaricides. However, the emergence of acaricide resistance has stimulated the search for new control alternatives, including phytocompounds with acaricidal and insecticidal potential. The aim of this study was to evaluate the acaricidal potential of Lavandula dentata essential oil against the engorged females of R. (B.) microplus. METHODS: Engorged females were obtained from infested bovines in dairy farms in Pernambuco, Brazil. L. dentata essential oil was extracted, and adult immersion test assays were performed using the following oil concentrations: 0.2%, 0.4%, 0.6%, 0.8%, and 1%. RESULTS: L. dentata essential oil at a concentration of 1% was lethal to all engorged females, and concentrations of 0.6% and 0.8% caused mortality of 98.6% and 99.1%, respectively. These concentrations disrupted the reproductive capacity of engorged females, reducing oviposition by more than 90% and preventing egg hatching by over 87%. CONCLUSION: The data revealed that L. dentata essential oil possesses effective pharmacological properties against R. (B.) microplus and could be used for tick control following in vivo evaluation, thus contributing to mitigating the negative impacts of synthetic acaricide use.

Short curing time bulk fill composite systems: volumetric shrinkage, degree of conversion and Vickers hardness.

This study aimed to evaluate volumetric polymerization shrinkage, degree of conversion and Vickers hardness of four bulk-fill resin composites light-activated with their dedicated light curing units (LCUs). Four groups were evaluated, according to the type of composite and curing mode: Tetric EvoCeram Bulk-fill (TEBO) and Tetric EvoFlow Bulk-fill (TEBF) were light-activated with Bluephase Style 20i (20s, in high-mode), while Tetric Powerfill (TEPO) and Tetric Powerflow (TEPF) were light-activated with Bluephase PowerCure (3s). Volumetric polymerization shrinkage test (n = 6) was performed in standardized box-shaped class-I cavities of extracted third molars (4 x 4 x 4 mm). Teeth were scanned before and after resin composite application by micro-computed tomography, and acquired data were evaluated with Amira software. Degree of conversion (n = 5) was evaluated at the top and bottom surfaces of composite cylindric samples (4 mm diameter, 4 mm thickness) using an FT-IR spectrometer (spectra between 1,500 and 1,800 cm-1, 40 scans at a resolution of 4 cm-1). Three Vickers indentations (50 g / 15 s), spaced 500 µm apart, were performed on the top and bottom composite surfaces and averaged. One-way ANOVA was used for data evaluation. TEPF showed the lowest volumetric polymerization shrinkage (p < 0.05), while the other composites were not significantly different within each other (p > 0.05). All materials presented a significant decrease in degree of conversion and Vickers hardness when compared top to bottom surfaces (p < 0.05). Bottom to top surface ratios for degree of conversion ranged from 0.8 (TEBO and TEPO) to 0.9 (TEBF and TEPF), and from 0.4 (TEPO) to 0.7 (TEBF and TEPF) for hardness. In conclusion, resinous materials present a decrease in hardness and degree of conversion from top to bottom even when a higher power is used, while the flowable material TEPF showed the lowest volumetric shrinkage values compared to the other materials.

Human Chorionic Gonadotrophin (hCG) induces changes in IFN-pathway and Interferon-Stimulated Genes (ISGs) on the bovine endometrium at Day 18 of pregnancy.

We hypothesized that the hCG modulates the expression of IFNT-pathway and ISGs in bovine endometrium during early pregnancy. The aim of the current study is to evaluate the effect of hCG on IFNT-pathway signals and ISGs expression in endometrial cells. For this, 29 non-lactating cross-bread cows were used in the study and submitted to a 9-day fixed-time artificial insemination (FTAI) protocol. The day of the AI was considered Day 0 (D0), and five days (D5) after the FTAI, the cows were allocated into two groups: Control and hCG group, when a hCG group received a single dose of 2.500UI of hCG. On day 18 after FTAI (D18) cows were slaughtered and endometrial tissue samples were collected. There was no difference between the embryo recovery rate of the cows in C compared to the hCG. The hCG group increased the accessory corpus luteum formation rate. The hCG resulted in greater serum progesterone concentration in the hCG group compared to the C on Day 14. Only the expression of IFNAR2 and STAT1 were upregulated on pregnant cows of the hCG group compared to the C group. The pathway genes (JAK1, STAT2, and IRF9) were not regulated. The mRNA abundance of ISG15, MX1, MX2, and OAS1 was upregulated in pregnant cows for hCG group, compared to C group. The results show that the administration of hCG, 5 days after AI, in addition to increasing the serum progesterone, modulates the expression of IFNT-pathway and ISGs on bovine endometrium on Day 18 of pregnancy.

The effects of a large-scale home visiting programme for child development on use of health services in Brazil.

Partnership between early childhood development interventions and primary health care services can help catalyse health care uptake by socially vulnerable families. This study aimed to assess the real-life effects of a large-scale home visiting programme [Primeira Infância Melhor (PIM)] in Brazil on the use of preventive (prenatal visits, well child visits, dentist visits and vaccination) and recovery (emergency room visits and hospitalization) health services. A quasi-experiment nested in a population-based birth cohort study was conducted. The intervention group was firstly defined as all children enrolled in PIM up to age 6 months, and afterwards stratified between those enrolled during pregnancy or after birth up to 6 months. Children receiving PIM were matched with controls on propensity scores based on 27 confounders to estimate effects on health service use from prenatal to age 2 years. Double adjustment was applied in outcome Quasi-Poisson regressions. No evidence was found for effects of PIM starting anytime up to 6 months (262 pairs), or for the children enrolled only after birth (133 pairs), on outcomes occurring after age 6 months. When the programme started during pregnancy (129 pairs), there was a 13% higher prevalence of adequate prenatal visits (prevalence ratio = 1.13; 95% confidence interval 1.01-1.27), but no effect on use of any other health service. Sensitivity analyses suggested longer participation in the programme with reduced visitor turnover might improve its impact on prenatal visits. Integration between PIM and primary health care was not adequate to affect overall patterns of contacts with health services. Nevertheless, prenatal home visits showed potential to increase health service contact during a sensitive period of development, indicating the need to start such programmes before birth, when there is more time for maternal care, and family engagement in a network of services is facilitated.

Effects of H2O2 pretreatment on the elemental fingerprints of bivalve shells and their implications for the traceability of geographic origin.

The fraudulent mislabelling of seafood geographic origin has been growing due to complex supply chains and growing consumer demand. To address this issue, seafood traceability tools, such as those based on elemental fingerprints (EF) of bivalve shells, have been successfully used to confirm their harvesting location. However, despite the usefulness of these methodologies, there is still room for optimization. Therefore, this study evaluated the effects of a routine procedure during bivalve shells preparation for ICP-MS analysis - their pretreatment with H2O2 to remove organic components. More specifically, the present study evaluated the effects of H2O2 on i) the elemental fingerprints of shells of two bivalve species (Ruditapes philippinarum and Cerastoderma edule) from four different locations over the north-western and the western Iberian coast, and ii) their influence on the accuracy of models (based on the EF of shells) used to confirm the geographic origin of these species. Significant differences were observed between untreated and pretreated shells of R. philippinarum (p within location ranging from 0.0001 to 0.0011) and C. edule (p ranging from 0.0001 to 0.0007 for C. edule) for both their elemental fingerprints as a whole and several individual elements. The accuracy of the models employed to determine the origin of the two bivalve species, using i) untreated shells, ii) pretreated shells, and iii) both pretreated and untreated shells grouped per location, was high, with the models accurately predicting the geographic origin of 100, 90 and 95% of R. philippinarum and 95, 100 and 95% of C. edule, respectively. These results show that the shifts in the EF of bivalve shells promoted by treating them with H2O2 prior to ICP-MS analysis did not affect the accuracy of the models used to confirm the geographic origin of both bivalve species. Therefore, the need to pre-treat bivalve shells with H2O2 can be dismissed in future studies addressing the traceability of bivalves when using ICP-MS, thus contributing to reducing environmental impacts and economic costs associated with this procedure, as well as the time required to obtain results.

Influence of Specimen Dimension, Water Immersion Protocol, and Surface Roughness on Water Sorption and Solubility of Resin-Based Restorative Materials.

The evaluation of water sorption and solubility is pivotal for the development of new resin-based restorative materials with the potential for clinical application. The purpose of the present study was to evaluate the influence of the specimen dimension, water immersion protocol, and surface roughness on the water sorption and solubility of three resin-based restorative materials. Disk-shaped specimens of 15 mm × 1 mm, 10 mm × 1 mm, and 6 mm × 1 mm were produced with a composite resin (Z100), a resin cement (RelyX ARC), and an adhesive system (Single Bond 2-SB2). The specimens were immersed in distilled water according to four protocols: ISO (all the specimens for each group were vertically immersed in 50 mL); IV-10 (the specimens were individually and vertically immersed in 10 mL); IH-10 (the specimens were individually and horizontally immersed in 10 mL); and IH-2 (the specimens were individually and horizontally immersed in 2 mL). The surface roughness (Sa and Sp) was evaluated using an atomic force microscope, and the degree of conversion was determined using FT-IR spectrometry. The specimen dimension and water immersion protocol had no effect on water sorption and solubility. For the three resin-based restorative materials, Sp was higher than Sa. The degree of conversion was not influenced by the specimen dimension. The variations in the specimen dimension and water immersion protocol compared to those determined by ISO 4049 did not prevent the comparison between the values of water sorption and solubility obtained for a given resin-based restorative material.

Pre and Post-high-intensity Interval Training Delays Colon Tumor Onset in a Syngeneic Mouse Model.

BACKGROUND/AIM: High-intensity interval training (HIIT) can trigger transient anti-tumor cytotoxicity through the mobilization of natural killer cells (NK cells) and myokines. Yet, the effects of HIIT on tumor development and microenvironment are unclear. MATERIALS AND METHODS: Male C57/BL6 mice were administered either MC38 of syngeneic colon cancer cells or vehicle in a single subcutaneous injection. Before injection, the training group completed four weeks of the HIIT program (progressive swimming training, 3/week, 10-12 min, 4-6% of body weight for overload). Following injection, trained mice continued to exercise for two additional weeks. RESULTS: Pre and post-HIIT training was effective in preventing tumor onset (p=0.0065), maintaining body weight gain, and counteracting splenomegaly by 40% compared to the tumor group. However, HIIT had no impact on suppressing tumor growth, modifying final tumor volume, or significantly changing tumor proliferation (Ki-67), connective tissue content, or DNA double-strand damage detected by phospho-histone gamma-H2AX (γ-H2AX). CONCLUSION: Pre and post-HIIT program is feasible for mice carrying a subcutaneous syngeneic tumor and effective in delaying tumor burden; however, HIIT did not alter colon tumor endpoints.

Pre-existing structural control on the recent Holuhraun eruptions along the Bárðarbunga spreading center, Iceland.

The active rift zones in Iceland provide unique insight into the geodynamic processes of divergent plate boundaries. The geodynamics of Iceland are studied intensively, particularly, by geophysical methods sensitive to active and/or visible structures such as earthquake seismic and Synthetic Aperture Radar observations or aerial photographs. However, older and less active structures, that may exert a strong control on the presently active geodynamics, are often buried beneath recent volcanic or sedimentary deposits and are-due to their passive mode-overseen by the typical geophysical investigations. Aeromagnetic surveys provide spatial information about subsurface magnetization contrasts relating to both active and inactive structures. However, the aeromagnetic data in Iceland were collected in the 1970-80s and are relevant only to large-scale regional rift studies. With the availability of reliable drones and light-weight atomic scalar sensors, high-quality drone magnetic surveys can provide an unprecedented spatial resolution of both active and passive structures of rift systems as compared to conventional airborne surveys. Here, we present the results of a drone-towed magnetic scalar field and scalar gradiometry study of the north-northeast trending Bárðarbunga spreading center to the north of the Vatnajökull ice cap, Iceland. Our results provide new information about the structural complexity of rift zones with evidence of densely-spaced, conjugate and oblique faults throughout the area. Evidence is shown of a hitherto unknown and prominent east-northeast trending fault structure that coincides with the northern tip of the main eruption edifice of the 1797 and 2014-15 Holuhraun volcanic events. We suggest that this pre-existing structure controlled the locus of vertical magma migration during the two Holuhraun events.

Diagnosis of Canine Visceral Leishmaniasis by Flow Cytometry Serology using the rMELEISH Multiepitope Antigen Coupled in a Functional Bead.

BACKGROUND: Visceral leishmaniasis (VL) is a zoonotic disease, with dogs being the main reservoir of the Leishmania infantum parasite. OBJECTIVE: To develop a new flow cytometry test to diagnosis canine VL (CVL) diagnosis. METHODS: The current study addresses a new flow cytometry test using beads coupled to the multiepitope antigen rMELEISH. RESULTS: In the study set of samples a sensitivity (87.1%) and specificity (89.9%) was observed. Considering the dogs' clinical status, 20/20 (100.0%) of the symptomatic sera tested positive, while 19/22 (86.4%) of the oligosymptomatic and 16/20 (80.0%) of asymptomatic were positive. In the non-infected control, all samples (0/30) tested as negative. In the cross-reaction control, the test was more efficient in dogs infected with L. braziliensis (2/10) and Trypanosoma cruzi (0/10), than those with Babesia canis (4/10) and Ehrlichia canis (4/10). Dogs immunized with different vaccines (Leishmune, Leish-Tec®, or LBSap) did not present serological reactivity. CONCLUSION: The flow cytometry serology through coupling the antigen rMELEISH in functional beads showed high accuracy in diagnosing CVL.

Development of a new assay for quantification of parasite load of intracellular Leishmania sp. in macrophages using flow cytometry.

Finding novel methodologies that enhance the precision, agility, and standardization of drug discovery is crucial for studying leishmaniasis. The slide count is the technique most used to assess the leishmanicidal effect of a given drug in vitro. Despite being consolidated in the scientific environment, it presents several difficulties in its execution, assessment, and results. In addition to being laborious, this technique takes time, both for the preparation of the material for analysis and for the counting itself. Our research group suggests a fresh approach to address this requirement, which involves utilizing nuclear labeling with propidium iodide and flow cytometry to determine the quantity of Leishmania sp. parasites present in macrophages in vitro. Our results show that the fluorescence of infected samples increases as the infection rate increases. Using Pearson's Correlation analysis, it was possible to establish a correlation coefficient (Pearson r = 0.9473) that was strongly positive, linear, and directly proportional to the fluorescence and infection rate variables. Thus, it is possible to infer a mathematical equation through linear regression to estimate the number of parasites in each sample using the Relative Fluorescence Units (RFU) values. This new methodology opens space for the possibility of using this methodological resource in the in vitro quantification of Leishmania in macrophages.