The SPCE/MWCNTs-COOH also exhibited satisfactory repeatability, reproducibility, and selectivity towards potential interferences. Moreover, for the first time, the electrochemical sensor allows determining the real concentrations of DF in environmental water samples without sample pretreatment steps.A simple, sensitive and time-saving differential-pulse adsorptive stripping voltammetric (DPAdSV) procedure using a screen-printed carbon electrode modified with carboxyl functionalized multiwalled carbon nanotubes (SPCE/MWCNTs-COOH) for the determination of diclofenac (DF) is presented. The sensor was characterized using optical profilometry, SEM, and cyclic voltammetry (CV). The use of carboxyl functionalized MWCNTs as a SPCE modifier improved the electron transfer process and the active surface area of sensor. Under optimum conditions, very sensitive results were obtained with a linear range of 0.1–10.0 nmol L Diclofenac (DF) is a nonsteroidal anti-inflammatory drug (NSAID), a derivative of aminophenylacetic acid with a strong anti-inflammatory, analgesic and antipyretic effect. The action of the drug causes the inhibition of inductive (COX-2) cyclooxygenases, responsible for the synthesis of proinflammatory prostaglandins in the site of inflammation and constitutive COX-1) and the synthesis of prostaglandins fulfilling a physiological function in the gastrointestinal tract and kidneys. DF is absorbed quickly and completely from the gastrointestinal tract and then eliminated completely within 12 h, approximately 60% in the urine and 33% in the faeces. DF is used as an analgesic and anti-inflammatory drug in rheumatoid arthritis, other connective tissue systemic diseases, gout attack, osteoarthritis, and the prevention and treatment of postoperative pain and neuralgia. DF is not recommended for children under 12 years of age and for people suffering from gastric and duodenal ulcer, aspirin-induced asthma, impaired hepatic function, renal insufficiency, and porphyria benzeneacetic acid sodium salt  was dissolved in deionized water to prepare a 0.01 mol L−1 NaAc–HAc buffer solution with a pH value of 4.0 ± 0.1. An accumulation potential (accE) of −0.25 V was applied when stirring the solution for a period of 60 s (accumulation time was represented by acct). After an equilibrium time of 5 s, DPAdSV curves were recorded from −0.25 to 1.5 V with an amplitude (A) of 125 mV, a modulation time (mt) of 10 ms, and a scan rate (ν) of 175 mV s−1. Then, the background curve was subtracted, and the DPAdSV curves were cut in the potential range of 0.4–0.8 V. The DPAdSV curves for each concentration of DF were recorded 3 times, and the average values of peak currents are shown with an SD for n = 3.Differential-pulse adsorptive stripping voltammetric (DPAdSV) measurements of DF under optimized conditions were carried out in a 0.1 mol Lv/v: 6:4). The flow rate of mobile phase was 1.0 mL min−1, and the temperature of thermostat was set at 25 °C. Injection volumes were 20 and 80 µL. All samples were analyzed in triplicate at a wavelength of 276 nm.Chromatographic conditions were based on the literature  with a sThe voltammetric and chromatographic methods were applied for the determination of DF in Vistula river (Poland) samples stored in sterile, polypropylene containers (Merck), which were collected from two places: at the sewage outlet (sample #1) and about 5 kilometres from the outflow of sewage (sample #2). The water samples were filtered using a 0.45 µm Millipore membrane and then directly analyzed without performing any special sample pretreatment procedure.−1 were recorded in a 0.1 mol L−1 NaAc–HAc solution with a pH value of 5.0 ± 0.1 at the surface of a bare SPCE and the surface of an SPCE/MWCNTs-COOH. DF showed an oxidation peak at 412 mV with the bare SPCE. At the SPCE/MWCNTs-COOH, the oxidation peak appeared at 377 mV with considerable enhancement in peak current (−1 (correlation coefficient r = 0.9997) and 0.5–200.0 nmol L−1 (r = 0.9971) at the surfaces of the bare SPCE and the SPCE/MWCNTs-COOH, respectively. The sensitivity values of DF determination at the surfaces of the bare SPCE and the SPCE/MWCNTs-COOH are equal to 0.019 and 0.040 µA/nmol L−1, respectively. Compared with the SPCE, the commercially available SPCE/MWCNTs-COOH provided a higher sensitivity and a wider linear range, which was connected with the developed active surface of the sensor modified with carboxyl functionalized carbon nanotubes.In order to understand the use of SPCE/MWCNTs-COOH for the assay of DF, the DPAdSV curves in the DF concentration range of 0.5–200.0 nmol L current . The oxia: 1.12 and 1.30 µm for the SPCE and the SPCE/MWCNTs-COOH, respectively). This confirmed that the whole SPCE surface was covered with a thin layer of carboxyl functionalized MWCNTs, which is also visible in the SEM images 6]. −1, and pI) and the square root of scan rate (v1/2). Based on these results, peak-to-peak separations (ΔE) and relative peak separations (0)χ for a scan rate of 175 mV s−1 and active surface areas (sA) based on the entire range of scan rates were calculated [The electrochemical properties of the SPCE and the SPCE/MWCNTs-COOH were also tested using cyclic voltammetry (CV) in a solution of 0.1 mol Llculated . The resThe above described results confirmed the benefits of using the SPCE/MWCNTs-COOH. Therefore, in further experiments, this kind of modified sensor was used for the analysis of DF. −1 DF were examined using a 0.1 mol L−1 solution of H2SO4, CH3COOH and CH3COONa + CH3COOH (NaAc–HAc) with pH values of 3.5 ± 0.1, 4.0 ± 0.1, 4.2 ± 0.1, 4.5 ± 0.1, 5.0 ± 0.1, and 5.6 ± 0.1, K2HPO4 + KH2PO4 with a pH value of 7.0 ± 0.1, (NH4)2SO4 + NH4OH with a pH value 8.3 ± 0.1, NH4Cl + NH4OH with a pH value of 10.0 ± 0.1, and NaOH with a pH value of 13.0 ± 0.1; the corresponding data are listed in a of 4.15, so for pH smaller than 4.15 the neutral species predominates, whereas at a greater pH the anionic species predominates [The effects of the type and the pH of the supporting electrolyte on the voltammetric responses of 0.05 and 0.1 µmol Lominates . Conside−1 (−1) signals were attained at a 0.1 mol L−1 concentration of the NaAc–HAc buffer solution with a pH value of 4.0 ± 0.1; hence, it was adopted for subsequent experiments.Furthermore, its concentration was evaluated from 0.05 to 0.5 mol L−1 B. The hi−1 NaAc–HAc buffer solution with a pH value of 4.0 ± 0.1 containing 1.0 µmol L−1 DF using v equal to 175 mV s−1 (CV curves (first and second cycles) were recorded in the 0.1 mol L5 mV s−1 A. DF was5 mV s−1 A. Theref−1 DF in the 0.1 mol L−1 NaAc–HAc buffer solution with a pH value of 4.0 ± 0.1 for v equal to 5–250 mV s−1 were observed. v of 50, 100, and 175 mV s−1. The oxidation peak potential shifted toward more positive values with the increase of scan rate, which confirmed that DF was irreversibly oxidized. The linear relationship between the DF peak current (Ip) and the square root of scan rate (v1/2) (Ip vs. logv (Ep) vs. log(v) plot  plot E using tl scheme F. accE), accumulation time (acct), amplitude (A), modulation time (mt), and scan rate (ν) on the DF oxidation peak current (0.01 and 0.05 µmol L−1) were studied. In order to find the optimum conditions for the DF determination at the surface of the SPCE/MWCNTs-COOH, the influences of various DPAdSV parameters including accumulation potential . The highest signals of DF for both studied concentrations were recorded at A equal to 125 mV (ν (50–200 mV s−1) on DF peak current at the constant values of A (125 mV) and mt (40 ms) was tested. ν of 175 mV s−1. Furthermore, mt was varied from 2 to 60 ms (for A of 125 mV and ν of 175 mV s−1). It was found that, for mt of 10 ms, the highest signals of DF were obtained  were 0.028 and 0.094 nmol L−1, respectively. The LOD and LOQ values demonstrated that the SPCE/MWCNTs-COOH can be used for analysis of environmental water samples, in which the DF concentration is in the range of 0.037–14.0 nmol L−1 [Under optimized conditions , the oxidation current responses were found to be proportional to the DF concentrations over the range of 0.1–10.0 nmol L−1 DF with 10 replicates and replicated on five different days, respectively. The results of the intra-day and inter-day precisions were 0.7% and 2.1%, respectively, indicating the satisfactory precious repeatability of the signal at the SPCE/MWCNTs-COOH surface. The reproducibility of the SPCE/MWCNTs-COOH was estimated by recording DPAdSV curves in the solution containing 10.0 nmol L−1 DF using three commercially available electrodes. The relative standard deviation (RSD) was calculated as 2.9% (n = 9), confirming the acceptable reproducibility of the proposed sensor.Moreover, the intra-day and inter-day precisions were verified for the determination of 10.0 nmol L−1 DF. It was noticed that ibuprofen (up to 2000-fold excess), caffeine (up to 2000-fold excess), paracetamol (up to 1000-fold excess), Ni2+ ions (up to 1000-fold excess), Fe3+ ions (up to 1000-fold excess), Zn2+ ions (up to 500-fold excess), Pb2+ ions (up to 500-fold excess), Sb3+ ions (up to 500-fold excess), Cu2+ ions (up to 100-fold excess), Cd2+ ions (up to 500-fold excess), V5+ ions (up to 100-fold excess), and Mo6+ ions (up to 100-fold excess) had negligible effects  on the assay of DF. Furthermore, the tolerance limits for the studied surfactants and humic acid were 5.0 mg L−1 for Triton X-100, 1.0 mg L−1 for CTAB, 2.0 mg L−1 for SDS, and 10.0 mg L−1 for humic acid . The results confirmed that the proposed procedure offers good selectivity for the determination of DF and the analysis of environmental water samples can by performed without sample preparation.In order to confirm the selectivity of the SPCE/MWCNTs-COOH, the DPAdSV responses of DF in the presence of different interferences found in environmental water samples were recorded. The tolerance limit was defined as the concentration, which gave an error of ≤10% in the determination of 10.0 nmol L−1 in Vistula river water sample #1 without sample pretreatment steps  and about 5 kilometres from the outflow of sewage (sample #2), were analyzed. nt steps . In sampIn order to test the accuracy of the DPAdSV method, samples were spiked with a standard solution of DF. The recovery values attained by the DPAdSV method were between 99.6% and 100.9%, which corresponded to the satisfactory degree of accuracy.−1, which was significantly above the real DF concentration in water samples. In order to determine lower concentrations of DF by the chromatographic method, an additional sample preparation step was required. According to the Student’s t-test, there were no significant differences between DF concentrations obtained by both methods. The calculated t values (expt) were 0.48 and 0.60, which were below the critical value equal to 1.81  and 95% confidence level). All these results indicated that the DPAdSV procedure using an SPCE/MWCNTs-COOH is highly selective and excellent for the determination of DF in real applications. The results with the DPAdSV method were compared with those obtained by the chromatographic method, HPLC/PAD. As can be seen in −1 and an LOD value of 0.028 nmol L−1. Furthermore, the SPCE/MWCNTs-COOH also showed satisfactory repeatability, reproducibility, and selectivity towards potential interferences. It should be clearly stressed that, for the first time, the electrochemical sensor was used for the determination of a real concentration of DF (0.42 ± 0.08 nmol L−1) in environmental water samples (Vistula river samples) without sample pretreatment steps. All these discussion indicated that the DPAdSV procedure using an SPCE/MWCNTs-COOH has great potential towards the determination of DF in real water samples to maintain environmental protection. Moreover, the DPAdSV procedure using an SPCE/MWCNTs-COOH can be applied in the DF field analysis.In summary, a simple, sensitive and time-saving DPAdSV procedure using an SPCE/MWCNTs-COOH was presented and successfully applied for the determination of DF. The modified sensor showed an improved sensing activity towards DF compared to a bare SPCE due to the effect of the modifier. The use of carboxyl functionalized MWCNTs improved the electron transfer process and the active surface area of the sensor. The SPCE/MWCNTs-COOH exhibited excellent current responses towards DF determination in the linear range of 0.1–10.0 nmol L


Doxorubicin (DOX), a chemotherapeutic drug widely used in the clinical setting, is known to cause serious cardiotoxicity and greatly reduces the survival rate as well as quality of life of patients receiving chemotherapy. Peroxisome proliferation activated receptor α (PPARα) is a type of ligand activated receptor of the nuclear hormone receptor family that regulates multiple gene expression. Several studies have shown that PPARα has anti-apoptotic and cardio-protective effects. However, its role in DOX-induced cardiotoxicity is rarely reported. In this study, we observed decreased expression of PPARα in the heart of tumor-bearing mice already treated with DOX; however, no such phenomenon was observed in tumor tissues. Next, we observed that the PPARα agonist, fenofibrate (FENO), had no effect on tumor progression; however, it enhanced cardiac function in tumor-bearing mice treated with DOX. Subsequently, recombinant adeno-associated virus serotype 9 (rAAV9) was used to manipulate the expression of PPARα in the heart of DOX-induced mice. Our results showed that PPARα gene delivery reduced cardiac dysfunction and mitochondria-dependent apoptosis in DOX-induced mice. Furthermore, we found that PPARα directly regulated the expression of mesenchyme homeobox 1 (MEOX1). Most importantly, the cardioprotective effects of PPARα could be neutralized by knocking down MEOX1. In summary, PPARα plays a vital role in DOX-induced cardiotoxicity and is a promising treatment target. Streptomyces mutants, is a widely used chemotherapeutic drug. It has a high affinity for DNA and forms DOX-DNA complexes, resulting in mitotic disorder and DNA double strand breakage , originally obtained from breakage . DOX is breakage . DOX is ectively . The inc00 mg/m2 . One of 00 mg/m2 . This pr00 mg/m2 . It is w00 mg/m2 . HoweverPeroxisome proliferation activated receptor α (PPARα), a kind of nuclear hormone receptor and regarded as a transcription factor, is highly expressed in the heart . Fatty aHowever, the role of PPARα in DOX-induced cardiotoxicity has not yet been fully clarified. In the present study, we demonstrated that the cardioprotective effect of PPARα was achieved by reducing oxidative stress and mitochondria-dependent apoptosis.2. When cells reached about 80% confluence, they were treated with a corresponding concentration of DOX. After 24 h, the cells were collected and used for follow-up detection.H9C2 cells, HEK293T cells, A549 cells, MDA-MB-231 cells, and TC-1 cells were obtained from the ATCC, and were cultured in 10% FBS Dulbecco’s modified Eagle’s medium (DMEM) at 37°C, with 5% COThe animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals, published by the United States National Institutes of Health . All experimental procedures were approved by the Experimental Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.in vivo, we used the mouse tumor cell line, TC-1 cells, to carry out subcutaneous implanting in 6- to 8-week-old male C57BL/6 mice. TC-1 cells (1 ×  106) were injected subcutaneously into each mouse. Tumor size was uniform among the groups at day 1. Subsequently, the mice were randomly divided into four groups: Control, FENO , DOX , and DOX+FENO. Tumor size was measured once every two days with a digital calliper. The shape of tumors was regarded as an elliptical sphere, and their volume were calculated according to the following formula: V = (width)2 × length × π/6. When the tumor was 2 cm in any dimension, the mice would be euthanized.To observe the effect of PPARα activation on tumor growth 11 vector genomes per mouse) was injected into the tail veins and DOX treatment  were randomly divided into six groups: Control, rAAV9-GFP, rAAV9-PPARα, DOX , DOX+rAAV9-GFP and DOX+rAAV9-PPARα. Then, rAAV9 , according to the manufacturer’s instructions and reverse-transcribed by PrimeScript RT reagent Kit with gDNA Eraser . RT-PCR was performed to quantify mRNA levels using TB Green Premix Ex Taq II . The primers used in this study are listed in For the detection of apoptosis, Annexin V-FITC/PI apoptosis detection kit  was used. All operations referred to the instructions of the manufacturer. Finally, quantitative analysis was performed by a FACStar Plus flow cytometer .2, for 24 h, the cells were treated using DOX, supplemented with or without Wy-14643. After incubating at 37°C in the dark for 1 h, absorbance was detected by enzyme-labeled instrument at 450 nm.Cell counting kit-8  was used to detect cell viability, in accordance with the protocol provided by the manufacturer. Briefly, logarithmic phase cells were collected followed by adjusting the cell number. Then, approximately 10,000 cells were added to each well of the plate. After culturing at 37°C, with 5% CORAAV9 system, which was a present from Dr. Xiao (University of North Carolina at Chapel Hill), was used as the vector to manipulate the expression of PPARα in the heart. We co-transfected the recombinant plasmid containing mouse PPARα or enhanced green fluorescent protein (EGFP) with the packaging plasmid into HEK293T cells. The preparation and purification of the virus has been described previously .After isoflurane (1–2%) anesthesia in mice, high-resolution probe and ultrasound imaging system  were used to detect the long-axis section and the short-axis section of the heart, respectively. Left ventricular hemodynamics was performed by Millar Catheter System, as described previously .The heart was fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm sections. Subsequently, H&E staining and Masson′s trichrome staining were performed to assess the myocardial cross-sectional area and fibrosis area using Image Pro Plus .DHE was used to detect ROS content in fresh frozen LV sections (6 μm thick), as described previously . The secTotal genomic DNA was extracted by TIANamp Genomic DNA Kit . The relative expression of mtDNA was detected by RT-PCR using TB Green Premix Ex Taq II, as described in previous studies .ATP content was measured by the ATP bioluminescent assay kit , according to the manufacturer′s instruction. The chemiluminescence was detected by a luminometer .All procedures were carried out in accordance with the instructions of In Situ Cell Death Detection Kit . 4′,6-diamidino-2-phenylindole (DAPI) was used to recognize the nuclei. After staining, a fluorescence microscope was used to observe the ratio of TUNEL positive cells.Detection of ROS in H9C2 cells was performed using 2′,7′-dichlorofluorescein diacetate . All procedures were carried out in accordance with the manufacturer’s standard instructions. Briefly, the cells were incubated with DCFH-DA, which was diluted in a 1: 1000 ratio with serum-free medium, in the dark at 37°C for 40 min. Finally, the cells were washed three times with serum-free cell culture medium, followed by detection by flow cytometry.To determine mitochondrial transmembrane potential in H9C2 cells, 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide  was used. Detailed procedure can be found in the protocol of the product. In brief, H9C2 cells were incubated with JC-1 staining buffer for 30 min in the dark at 37°C. Then, the cells were rinsed twice with JC-1 rinsing buffer at 4°C. Finally, the cells were resuspended with phosphate-buffered saline. The fluorescence intensity was measured by flow cytometry.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81448), GSE59672 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE59672), and GSE23598 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE23598). These data sets contained global gene expression profiles in the hearts of control and DOX-induced mice. Intersectional analyses were performed using R project (v3.4.1). The finding criterion of differentially expressed genes was according to p value < 0.05. Genes in the DOX treatment group that changed more than twice as much as those in the control group were selected. Subsequently, the genes with different expression were screened by intersectional analysis. Bioinformatics websites PROMO  and LASAGNA (http://biogrid-lasagna.engr.uconn.edu/lasagna_search/) were used for predicting differentially expressed genes that may be regulated by PPARα  was inserted into the plasmid. In addition, homologous PPARα cDNA was inserted into PCDNA3.1 expression vector. These were co-transfected into HEK293T and H9C2 cells, respectively, to carry out luciferase reporter assay 48 h after transfection, using Renilla luciferase activity as the reference. Each reporter was repeated in at least three independent experiments.For ChIP assay, ChIP Assay Kit  and PCR purification Kit  were used. Primer sequences were as follows: #1 (−1734 to −1724 bp) F: 5′ CCTGGGCGACAGAACGAGAC 3′, R: 5′ GGGGTTTGAGGTTAGACGGGT 3′, and #2 (−1091 to −1081 bp) F: 5′ CTTTGGAAGCAGCCAGCAG 3′, R: 5′ GCAATTTCTTGGAGGACGACA 3′. Anti- PPARα was from Abcam , and IGg was from Cell Signaling Technology . All the procedures were the same, as described previously .H9C2 cells were transfected with Si-MEOX1  or Si-NC using Lipofectamine 2000 Transfection Reagent , according to the manufacturer′s instruction. Forty-eight hours later, the cells were pre-treated with or without Wy-14643 for 1 h, followed by DOX treatment for an additional 24 h. The cells were then collected for follow-up detection.All data are represented as means ± SEM. Unpaired two-tailed Student’s t test for independent data of normal distribution and one-way ANOVA with Tukey’s test for multiple comparisons were performed in GraphPad Prism 5.0 . Paired two-tailed t test was applied to ChIP assay. Statistical P values < 0.05 were considered significant.In this study, we detected the expression of PPARα in the heart and tumor tissues of tumor-bearing mice treated with DOX. We found that PPARα expression decreased in heart tissues  . Thus, Dvia various pathways. TOP2α is abundantly expressed in the tumor tissues; therefore, a combination of DOX and TOP2α is the molecular basis for eliminating tumor cells. Unfortunately, the expression of TOP2β is abundant in the heart, and the binding of DOX to TOP2β is related to cardiotoxicity , which can generate toxic and highly reactive hydroxyl radicals (OH∙) during the Fenton reaction, finally resulting in ROS generation. The resulting ROS can react with nearby mitochondrial biomolecules, including proteins, lipids, and nucleic acids. DOX can also react with mtDNA, forming a complex that interferes with normal mitochondrial function, protein expression, and lipid oxidation , resulting in a decrease in mitochondrial transmembrane potential, increase in mitochondrial membrane permeability, and release of apoptotic proteins from the mitochondria. Meanwhile, due to the oxidative damage of mtDNA, synthesis of mitochondrial ATP is inhibited, which leads to mitochondria-dependent apoptosis . Therefovia different mechanisms. Taken together, we have proved that PPARα can improve mitochondrial function and reduce DOX-induced cardiotoxicity by regulating the expression of MEOX1.Our research also has certain limitations: we could not determine how MEOX1 regulates mitochondrial function, and this needs to be confirmed by future studies. In addition, MEOX1 was previously shown to cause pressure overload-induced hypertrophy . We assuIn conclusion, our findings suggest that PPARα may be a potential target against DOX-induced cardiotoxicity.All datasets generated for this study are included in the article/The animal study was reviewed and approved by Experimental Animal Research Committee of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.LW originally designed the scientific research. WW performed experiments and data analyses and wrote the manuscript. YW monitored the project progression. ZZ, QF, and DW provided technical assistance. WW, QF, LW, and YW were responsible for revision of this manuscript. All authors contributed to the article and approved the submitted version.This study was supported by foundations from National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Glomerular filtration rate (GFR) is usually estimated from equations using serum creatinine (sCr), with adjustment for gender, age, and race (black or nonblack). The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) is the preferred equation for adults, but it was validated for the United States population. We intended to evaluate if the race-ethnicity adjustment proposed for the sCr-based CKD-EPI equations is appropriate for the Brazilian population. 51Cr-EDTA and used as the reference. We compared values of mGFR and estimated GFR (eGFR) by CKD-EPI equations based on sCr (eGFRCr) and on the combination of sCr and sCys (eGFRCr-Cys). For African Brazilian patients, eGFR was calculated either without or with race adjustment. Accuracy was considered acceptable if the difference between the values of eGFR and mGFR was ≤30% (P30).  CKD outpatients had blood samples collected for determination of sCr and serum cystatin C (sCys) levels. GFR was measured (mGFR) by plasma clearance of 2. Mean eGFRCr and eGFRCr-Cys without race adjustment were 47.8 ± 30.1 ml/min/1.73 m2 and 46.4 ± 30.3 ml/min/1.73 m2, respectively. The corresponding P30 accuracy values were 79.0% and 83.0%. In the African Brazilian subgroup, values for mean mGFR and eGFRCr either without or with race adjustment were 49.8 ± 32.2 ml/min/1.73 m2, 50.4 ± 32.7 ml/min/1.73 m2, and 58.4 ± 37.9 ml/min/1.73 m2 (P < 0.001 vs. mGFR), respectively. P30 accuracy values for eGFRCr either without or with race adjustment were 75.4% and 67.2%, respectively.  100 patients were enrolled . Mean mGFR was 46.7 ± 29.2 ml/min/1.73 m The use of CKD-EPI equations without race-ethnicity adjustment seems more appropriate for the Brazilian population. GFR can iohexol . However iohexol . DespiteThe sCr-based equations proposed by the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) for adults are used worldwide and include a race-ethnicity adjustment for African Americans . Such anOur study aimed to evaluate the appropriateness of the race-ethnicity adjustment of the sCr-based CKD-EPI equations for the Brazilian population.Cr) or combination of serum creatinine and serum cystatin C (eGFRCr-Cys) in Brazilian patients with chronic kidney disease.This is a cross-sectional study of the performance of CKD-EPI equations for estimation of GFR derived from serum creatinine .CKD-EPI equations for eGFRA specific form with demographic, anthropometric, and clinical data was fulfilled in the day of GFR measurement. The local ethics committee approved the study. All participants signed the informed consent form.All CKD patients older than 18 years registered at the Nephrology Division Clinics of the University Hospital were eligible to participate in this study. Exclusion criteria were pregnancy, liver cirrhosis, metastatic cancer, paraplegia, quadriplegia, or limb amputation.Participants were classified according to the three main race-ethnicity categories defined by the Brazilian Institute of Geography and Statistics: white, black, and mixed-race. The researcher in charge defined race-ethnicity according to the phenotypic appearance, on the day of GFR measurement. For this study, black and mixed-race subjects were classified into a single group and named as African Brazilians.51Cr-EDTA plasma clearance using the “dose × decay/interception” method described by Brochner-Motersen [2.The procedures for the assessment of GFR started around 8:00 a.m. at the Nuclear Medicine Unit of the University Hospital. GFR was determined by Motersen  and modiMotersen . GFR wasMotersen  and expr51Cr-EDTA  was injected in bolus (minute 0), and the cannula was flushed with saline and removed. Blood samples (10 mL) were collected in heparinized tubes from the contralateral upper limb peripheral vein at 150, 195, and 240 minutes for patients with estimated GFR ≥ 30 mL/min/1.73 m2 and 150, 195, 240, and 480 minutes for those with GFR estimated below such threshold. To define whether the duration of the evaluation would be 240 or 480 minutes, GFR was estimated by the CKD-EPI equation using the last sCr value available in the medical record.First, a cannula was inserted into a forearm vein, and a 10 mL blood sample was obtained for sCr and sCys measurement. Next, 51Cr-EDTA counting. Radioisotopic counting lasted 30 minutes on the gamma counter . The value for each point was defined by the average of the two aliquots counting.Blood samples were centrifuged for 10 minutes at 3000 rpm and separated into two 2 mL plasma aliquots for sCr was measured in the Roche/Hitachi Cobas© system by the Jaffé method traceable to isotope dilution mass spectroscopy . sCys was measured in the Siemens BN-II© analyser by nephelometry  and calibrated to the International Federation of Clinical Chemistry Working Group for Standardization of Serum Cystatin C (ERM-DA471/IFCC reference).2; stage 2, 89 to 60 mL/min/1.73 m2; stage 3a, 59 to 45 mL/min/1.73 m2; stage 3b, 44 to 30 mL/min/1.73 m2; stage 4, 29 to 15 mL/min/1.73 m2; and stage 5, <15 mL/min/1.73 m2.CKD staging based on GFR was defined according the Kidney Disease: Improving Global Outcomes (KDIGO) classification : stage 1We also assessed the ability of each equation to correctly classify the patients in the same CKD stage defined by the measured GFR and expressed as the percentage of concordance.Bias  = estimated GFR − measured GFRPrecision = median and interquartile interval of the difference between estimated GFR and measured GFRAccuracy (P30) = percentage of estimated GFR values with a difference equal to or lower than 30% of measured GFREquations for GFR estimation had their performance analysed according to bias, precision, and accuracy:Differences between measured and estimated GFR were analysed by the Bland–Altman method and expressed in graphs, with measured GFR on the abscissa and the difference (measured − estimated GFR) on the ordinate, in which negative values indicate GFR overestimation by the equation, whereas positive values mean underestimation.Normality of data distribution was tested using the Kolmogorov–Smirnov test. Continuous variables are expressed as mean ± standard deviation in case of a normal distribution or as median (interquartile range), otherwise. Categorical variables are shown as frequencies.t-test. Frequencies were compared by Fisher's exact test. P values <0.05 were considered significant.Comparisons between the means were made by paired Analyses were performed using SPSS® software, version 18.0 for Windows .The general characteristics of the 100 participants are presented in 2. The eGFRCr with no race-ethnicity adjustment was 47.8 ± 30.1 ml/min/1.73 m2 (P=0.38 vs. mGFR), whereas eGFRCr including race-ethnicity adjustment was 52.7 ± 34.9 ml/min/1.73 m2 (P < 0.0001 vs. mGFR). The eGFRCr-Cys without race-ethnicity adjustment was 46.4 ± 30.3 ml/min/1.73 m2 (P=0.70 vs. mGFR), whereas eGFRCr-Cys including race-ethnicity adjustment was 48.8 ± 32.2 ml/min/1.73 m2 (P=0.54 vs. mGFR), In the whole group, mGFR was 46.7 ± 29.2 ml/min/1.73 m2. The eGFRCr was 43.7 ± 27.7 ml/min/1.73 m2 (P=0.26 vs. mGFR), and eGFRCr-Cys was 41.6 ± 27.4 ml/min/1.73 m2 (P=0.85 vs. mGFR)  .2, and it was not significantly different from the white participants. The eGFRCr without race-ethnicity adjustment was 50.4 ± 32.7 ml/min/1.73 m2 (P=0.74 vs. mGFR), whereas eGFRCr with race-ethnicity adjustment was 58.4 ± 37.9 ml/min/1.73 m2 (P < 0.001 vs. mGFR). The eGFRCr-Cys without race-ethnicity adjustment was 49.4 ± 31.8 ml/min/1.73 m2 (P=0.74 vs. mGFR), whereas eGFRCr-Cys adding race-ethnicity adjustment was 53.4 ± 34.4 ml/min/1.73 m2 (P=0.012 vs. mGFR)  .2) tended to be higher among whites   .Cr with race-ethnicity adjustment to 66.7% for whites using eGFRCr-Cys equation, without statistically significant difference  of eGFRCr equation without race-ethnicity adjustment for all patients, whites, and African Brazilians were 0.1, 0.7 and −0.5, respectively. The correspondent bias (mL/min/1.73 m2) of the eGFRCr-Cys equation without race-ethnicity adjustment was −0.1, 0.4, and −0.2. After the inclusion of race-ethnicity adjustment, there was a significant increase in the bias of both eGFRCr and eGFRCr-Cys equations for all participants and the subgroup of African Brazilians , age, gender, body mass index, and body surface area. They found that CKD-EPI equation with the African American race-ethnicity correction factor overestimated GFR in African Europeans. Nonetheless, African Europeans had serum creatinine 8% higher than their matched white Europeans, suggesting that a lower race-ethnicity correction is needed. In a recent study, Bukabau et al. [2.Firstly, Levey et al. , in 1999u et al.  analysedIn a previous study conducted in São Paulo, Brazil, the creatinine-based MDRD and CKD-EPI equations, with race-ethnicity and no race-ethnicity adjustment, were compared to the measured GFR by iohexol plasma clearance in 244 patients followed at a glomerulopathy outpatient clinic. The authors have found no increase in accuracy by adding race-ethnicity adjustment in the equations. However, only 8% of patients were African Brazilians .Our finding of more unsatisfactory performance of equations by adding race-ethnicity adjustment indeed did not come as a surprise for us. In a previous study, our group analysed 1,303 individuals living in the city of Niterói, Rio de Janeiro, and 33% self-classified as white, 41% as brown, and 26% as black. In that analysis, we found no differences in serum creatinine values between the race groups, even after stratification by sex and age ranges, raising the question of whether adding race-ethnicity adjustment to eGFR equations would be appropriate for our population. However, the absence of measured GFR weakened our conclusions . Those fIf the use of race-ethnicity adjustment had improved the performance of eGFR equations in our study, probably its routine adoption at the clinical practice in Brazil would not be an easy task. Brazilian population has an ethnic composition made up of an extensive mixture of three different ancestral roots: Europeans, sub-Saharan Africans and, to a lesser extent, Native Americans . This miQ), in which Q is the median sCr from healthy populations to account for gender and age. Thus, the FAS equation can be used indistinctly in children, adolescents, adults, and elderly, obviating the need of specific equations for each phase of life [2.The use of new equations that are not dependent on a race/ethnicity variable could solve this issue and be adopted worldwide and for different ethnical groups. The FAS equation seems a very promising alternative, since it is based on normalized SCr (sCr/ of life . Later,  of life . Even th of life , with 49Another critical issue that deserves discussion would be the necessity and convenience of developing specific eGFR equations for the Brazilian population. Our findings suggest that this would not be needed since the performance of the equations, with no race-ethnicity adjustment, was quite satisfactory, being just slightly more imperfect than that described in the original validation studies, with a little lower accuracy but no significant bias. The performance of creatinine-based CKD-EPI equations with no race-ethnicity adjustment was also similar to CKD-EPI equations using both sCr and sCys. Furthermore, the Brazilian population is substantially racially diverse, whereas countries in which specific equations were developed or adapted in order to improve the performance had a very homogeneous population, such as Japan . Thus, wOur study presents several limitations, including the small sample size and the geographical restriction of the studied population who probably is not representative of the whole Brazilian population.In conclusion, the performance of CKD-EPI equations is acceptably good and can be used in the Brazilian population, and no race-ethnicity adjustment in creatinine-based CKD-EPI equations seems necessary and could indeed worsen the performance of the equations. Additional studies enrolling a broader number of patients are desirable to confirm our findings.


Trypanosoma brucei rhodesiense. Trypanosoma congolense was the most prevalent Trypanosoma sp., which was found in Nkhotakota , followed by Kasungu . Of note, the prevalence of T. b. rhodesiense detected by SRA PCR was high in Kasungu and Nkhotakota showing 9.5% (19 of 199) and 2.7% (5 of 185), respectively. We report the presence of animal African trypanosomes and T. b. rhodesiense from cattle at the human–livestock–wildlife interface for the first time in Malawi. Our results confirmed that animal trypanosomes are important causes of anemia in cattle and that cattle are potential reservoirs for human African trypanosomiasis in Malawi.This study aimed to identify trypanosomes infecting cattle in Malawi in order to understand the importance of cattle in the transmission dynamics of Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). A total of 446 DNA samples from cattle blood from three regions of Malawi were screened for African trypanosomes by ITS1 PCR. The obtained amplicons were sequenced using a portable next-generation sequencer, MinION, for validation. Comparison of the results from ITS1 PCR and MinION sequencing showed that combining the two methods provided more accurate species identification than ITS1 PCR alone. Further PCR screening targeting the serum resistance-associated (SRA) gene was conducted to detect  Trypanosoma spp. and is mainly transmitted by tsetse flies [T. congolense, T. vivax, and T. brucei brucei [T. congolense and T. brucei, which are transmitted by tsetse flies, T. vivax can also be transmitted mechanically by other hematophagous flies; as a result, it has a broader geographical distribution [African trypanosomiasis is caused by protozoan parasites of se flies . This dise flies . Animal i brucei . Infectii brucei . AAT affi brucei , 50. Clii brucei . AAT remi brucei . Unlike ribution .T. b. gambiense and T. b. rhodesiense infections, respectively [T. b. gambiense is an anthroponotic disease that depends primarily on human-to-human transmission; humans act as the main reservoir, while animal reservoirs play a minor role [T. b. rhodesiense is a zoonotic disease that affects mainly animals (wildlife and livestock); humans are considered accidental hosts [Human African trypanosomiasis (HAT) or sleeping sickness occurs in two forms with different features, due to ectively . Gambiennor role . It is dnor role . Gambiennor role . In contal hosts . It is fal hosts . The disal hosts .T. b. rhodesiense [T. b. rhodesiense due to high exposure of humans to agriculture [T. b. rhodesiense [T. b. rhodesiense in Kenya [Control of rhodesiense HAT is challenging because animals act as reservoirs for disease transmission , 63. Altdesiense . Livestoiculture . In Ugandesiense , 62, andin Kenya  and Tanzin Kenya .T. b. rhodesiense infections, HAT in Malawi is characterized by the distinct clinical sign of chronic hemolymphatic stage infection without the formation of a chancre; this makes diagnosis difficult [T. congolense, T. vivax, and T. brucei in Malawi using an indirect AbELISA serological detection method [Trypanozoon species to the subspecies level  [T. b. rhodesiense by PCR has been widely conducted by targeting the human serum resistance-associated (SRA) gene [T. godfreyi and T. vivax because they share overlapping amplicon size ranges.In Malawi, rhodesiense HAT has been a burden for decades . Unlike ifficult , 29. Endifficult , 30, wheifficult , 48. A pn method . Howevern method , 58. In n method , 43. Beciperdum) , 43, detRA) gene . IdentifMinION, developed by Oxford Nanopore Technologies, is a portable next-generation sequencer (NGS) that connects to a laptop computer through a USB cable , 36. MinIn this study, MinION NGS was combined to PCR-based methods to determine the prevalence of human and animal trypanosomes in cattle and to understand their epidemiological importance for HAT and AAT in Malawi.The ethical clearance for animal sampling was obtained from the Ministry of Agriculture, Irrigation and Water Development in Malawi through the Department of Animal Health and Livestock Development with reference number 10/15/32/D.This is a descriptive, cross-sectional study of trypanosome infection in cattle, conducted by analyzing DNA material from 446 bovine blood samples. Samples were collected from cattle in three districts in Malawi: Kasungu, Nkhotakota, and Lilongwe, in February and March 2018 during the rainy season. Information on farmers and cattle populations was obtained from the District Agriculture Development Office (DADO) for each district through their District Animal Health and Livestock Development Officer (DAHLDO). The sampling map  was consT. b. rhodesiense. Both clinically healthy animals and animals with clinical signs were randomly selected within farms. In Kasungu and Nkhotakota districts, a total of 199 and 185 cattle blood samples were collected from 29 and 26 farms, respectively. These farms were selected on purpose from a farmer registry list (Supplementary Table 1), since the selected areas are situated in the human–livestock–wildlife interface area in the vicinity of Kasungu National Park and Nkhotakota Wildlife Reserve , following the manufacturer’s protocol.Before sampling, the venipuncture site was disinfected with a methylated spirit swab. Then, 5 mL of blood were drawn by venipuncture of the external jugular vein into vacutainer EDTA tubes. Packed cell volume (PCV) counting was conducted to determine anemia; animals with a PCV Supplementary Table 2). PCR was performed in a final volume of 25 μL, comprising 12.5 μL Ampdirect plus , 0.125 μL BIOTAQ HS DNA Polymerase (5 U/μL) , 0.625 μL of each 10 mM primer, 0.5 μL of 2% DMSO, 8.625 μL RNase-free water, and 2 μL extracted DNA. The temperature and cycling profile included initial denaturation at 95 °C for 10 min followed by 37 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 90 s, extension at 72 °C for 2 min, and final extension at 72 °C for 7 min. PCR products were examined by electrophoresis in 1.5% agarose S  in Tris-acetate EDTA (TAE) buffer and stained using GelRed  dye before being visualized under ultraviolet light in a transilluminator.DNA samples were subjected to PCR amplification of the rRNA ITS1 region to identify all the pathogenic African trypanosome species/subspecies. ITS1 PCR was carried out using the primers described by Gaithuma et al.  , a constant product of approximately 415–431 bp; for T. congolense, 560–705 bp; for T. simiae, 331–343 bp; for T. godfreyi, 220 bp; for T. theileri, 269–350 bp; and for T. vivax, 226–238 bp [T. congolense, T. brucei, T. vivax, T. godfreyi, and T. simiae [Signals on gel were judged by three investigators independently. They were classified as follows: positive (clear band), negative, and ambiguous (faint band or smearing). Trypanosome species were identified based on the size differences for members of the subgenus 6–238 bp . The pla. simiae  were comSupplementary Table 2), 0.5 μL DMSO 2%, 2.55 μL RNase-free water, and 2 μL extracted DNA. PCR conditions were as follows: an initial hold at 95 °C for 10 min, followed by 10 cycles of 94 °C for 30 s, 60 °C for 1 min, 72 °C for 2 min, and a final extension at 72 °C for 5 min. Amplicon of the index PCR was prepared for MinION-compatible DNA libraries using a Ligation Sequencing Kit 1D SQK-LSK109 and Native Barcoding Kit EXP-NBD103  as per their instruction manuals. The indexed amplicons were purified by AMPure XP , and equal concentrations of each sample were pooled together to obtain 12 for each pool. End repair and dA-tailing were performed to the pooled, barcoded amplicons using the NEBNext UltraII End Repair/dA-Tailing module  per the Oxford Nanopore 1D Native barcoding genomic DNA sequencing protocol. Subsequently, the purified, end-prepped DNA was ligated with the Adapter Mix (AMX) using the NEBNext Quick T4 DNA Ligase . Final adapted DNA libraries were purified, and the pre-sequencing mix was eluted and quantified. The flowcell, FLO-MIN107 , was primed for loading and sequencing of the library. Before sequencing, MinION and flowcell were assembled and connected to a computer. Platform QC was run using MinKNOW software.To prepare a MinION sequencing library, amplicons of the ITS1 PCR were used as template then indexes were added by additional PCR. The indexed amplicons were further ligated with adapter DNA provided by Oxford nanopore; then, a sequence-ready library was obtained. Positive, ambiguous samples, and a positive control from ITS1 PCR were sequenced as follows. For the indexing, reagents comprised 5 μL Ampdirect plus , 0.05 μL BIOTAQ HS DNA Polymerase (5 U/μL) , 0.2 μL of each 10 mM ITS1-index primers . De-indexing was performed using custom scripts. In brief, indexed primer sequences were aligned on each MinION read using LAST , and theT. b. rhodesiense using the primers described by Radwanska et al. [Supplementary Table 2). Reagents used for each reaction included 5 μL Ampdirect plus , 0.05 μL BIOTAQ HS DNA Polymerase (5 U/μL) , 0.2 μL of each 10 mM primer, 2.55 μL RNase-free water, and 2 μL extracted DNA. The temperature and cycling profile included an initial hold for 10 min at 95 °C, followed by 40 cycles at 94 °C for 30 s, 60 °C for 1 min, 72 °C for 1 min, and a final extension at 72 °C for 5 min. PCR products were examined by electrophoresis in 2% agarose S  in TAE buffer and stained using GelRed  dye before being visualized under ultraviolet light.SRA PCR was employed to identify a et al.  (Supplemp < 0.05).The chi-square test was used to analyze the relationship between the infection rates by animals, region, and anemic status  and one mixed infection with T. congolense and T. vivax .Forty-four positive, 8 ambiguous, and 394 negative samples were obtained by ITS1 PCR. For positive samples, we identified 21 amplicons with a single band size of 620–700 bp, indicating a single T. congolense, 24 samples (including single and mixed infections) were positive by ITS1 PCR, and T. congolense sequences were detected in the same 24 samples and one additional sample, A17, by the MinION system (Supplementary Table 3). For T. vivax, 10 samples were positive, and the same 10 samples were confirmed by sequencing, as well as two additional samples, D07 and E60. E60 was a mixed T. congolense and T. vivax infection. For T. theileri, two samples were positive in both ITS1 PCR and the MinION system.In total, 503,039 reads were obtained from 53 multiplexed ITS1 amplicons, including one positive control. Taxonomic identity was determined for 369,673 reads using a BLAST homology search. For Trypanozoon were identified by ITS1 PCR. However, using the MinION system, Trypanozoon sequences were detected in only 3 of these 11 products (Supplementary Table 3). Most cases, with the exception of A37, produced reads for Trypanozoon that were much lower than our threshold (50 reads minimum). For A37, 172 reads for Trypanozoon out of 2965 reads were obtained, but reads were below our criteria, with a minimum of 50 reads and more than 20% of total reads; therefore, this sample was negative even though we could not eliminate the possibility of actual Trypanozoon infection.In contrast, 11 samples positive for Supplementary Table 3). E60 showed a clear signal for Trypanozoon and a faint signal for T. godfreyi or T. vivax. However, the MinION system provided substantial numbers of sequences to show that the sample was a mixed infection of T. congolense and T. vivax (Supplementary Table 3). Another case, A37, appeared to be a mixed infection of T. congolense and Trypanozoon by ITS1 PCR; however, MinION results did not support Trypanozoon infection as described above.Three confirmed cases of mixed infections were observed  and Kasungu . Trypanosoma vivax was found in Nkhotakota . Non-pathogenic Trypanosoma species T. theileri  was also detected in Nkhotakota. There were three mixed infections: one T. congolense with T. b. brucei, and two T. congolense with T. vivax , followed by Nkhotakota   was positive when analyzed by the ITS1-MinION detection system (Supplementary Table 3). of 185) . The detp < 0.05).These trypanosome parasites were all detected in two districts, with the highest prevalence being in Nkhotakota , followed by Kasungu  , while np < 0.05)  infections. In contrast, of 430 non-anemic cattle, only 49 heads (11.4%) were positive for trypanosome infection. In particular, infection with T. congolense showed a significant difference in anemia status compared to other species (p < 0.05). A significant difference in the anemic status of T. congolense-infected cattle was observed in both single-infected cattle and mixed-infected cattle (There was a significant difference in the anemic status (PCV < 24%) between cattle with trypanosome infection and without infection ( < 0.05) . Of 16 ad cattle .Trypanosoma sp. by ITS1 PCR and sequencing analysis was largely consistent for T. congolense, T. theileri, and T. vivax. This suggests that MinION sequencing can detect and differentiate trypanosome species. In addition, MinION sequencing appreciably remedied four downsides of ITS1 PCR: ambiguous signals, similar amplicon sizes, nonspecific signals, and multiple infections. We successfully detected a substantial number of reads from samples with ambiguous ITS1 PCR amplicons. T. godfreyi and T. vivax were differentiated. On the other hand, nonspecific amplification hampers correct identification and can lead to false positives. We observed 11 signals of approximately 450 bp corresponding to Trypanozoon; however, most were not supported by sequencing. These could be annotated as false positives without sequencing validation. Apart from nonspecific signals, all samples definitively annotated by the ITS1 PCR had more sequence reads than the threshold of 50. Since we detected enough reads for other trypanosome species from ITS1 PCR, the discrepancy presumably derived from nonspecific PCR amplification of fragments of the same size as the Trypanozoon amplicon. Thus, in several cases, disagreement between ITS1-PCR gel analysis and ITS1-MinION sequence analysis was observed, likely due to nonspecific amplification and misjudgement of the ITS1-PCR gel analysis. Multiple infections were also successfully confirmed by sequencing. All these observations support the assertion that ITS1 PCR validated by sequencing analysis using MinION increases the reliability of parasite detection. Conversely, we obtained only tentative annotation in some samples, even though substantial sequence reads were acquired. Annotation depends on the threshold, and we applied a conservative value to eliminate false positives rather than allow false negatives. BLAST homology search and de-indexing steps were possible sources of error owing to the low accuracy of MinION sequencing. The effective range of ratios and absolute numbers of parasites in mixed infections should also be determined. These points should be clarified and optimized in future studies. The combined approach of ITS1 PCR and MinION has the potential to exclude false positives by nonspecific amplification and less-objective human decisions. Sequencing itself became cost-effective because of the multiplex sequencing system using MinION indexes and barcodes. The quality of field samples is occasionally poor and leads to unexpected nonspecific amplification, as observed in our cases. MinION provides sequence information that can exclude nonspecific amplification and thus improve specificity [In this study, we applied next-generation sequencing (NGS) technologies, particularly MinION, a field-friendly, portable, rapid, and affordable NGS device. ITS1 amplicons, including ambiguous signals, were subjected to sequencing analysis. Identification of cificity . TherefoTrypanozoon subspecies owing to the highly conserved sequence of the species [Trypanozoon especially for T. b. rhodesiense as reported before [T. b. rhodesiense in a more sensitive and specific manner.ITS1 PCR in combination with MinION sequencing provides sequence information to identify a broad range of trypanosome species in a more reliable manner compared to the conventional ITS1 PCR with gel analysis only . However species . In addi species , 49, or  species . In our T. congolense. This is in agreement with reports from other southern African countries [Trypanosoma congolense and T. vivax were more prevalent in Nkhotakota than in Kasungu, with no parasites seen in Lilongwe. Possible reasons for this trend may be the different methods applied for AAT control. Nkhotakota wildlife services have intensified blue cloth targets in the park to reduce the number of tsetse flies. In Kasungu, local veterinarians and farmers apply an anti-parasitic treatment, Berenil (diminazene aceturate), to the cattle twice every year before and after the herding season. Berenil is a common therapeutic and preventive medicine for animal trypanosomiasis that has been used for >60 years [T. vivax in cattle by biting flies. However, as T. vivax was detected in Nkhotakota, this emphasizes the necessity of sustained non-tsetse vector control for animal trypanosomiasis in the region. Also, since we collected a limited number of cattle samples only at three locations during short period, a broader countrywide survey is required to assess the overall AAT/HAT situation in the country.In this study, the most common trypanosome detected was ountries , 58. Try60 years . Since s60 years , 42. In 60 years . On the T. congolense infection tended to be anemic, as compared to those infected with other trypanosome species, suggesting the importance of controlling AAT.The PCV value of infected cattle was significantly lower than that of non-infected cattle. Anemia has long been considered the main clinical sign of trypanosomiasis in both humans and animals , 16. AneT. b. rhodesiense prevalence in tsetse flies is higher in Malawi, according to xenomonitoring data [T. b. rhodesiense in cattle has been suspected in these areas [Malawi is rich in ecosystems where humans, livestock, and wildlife populations exist close to each other. Compared with neighboring countries such as Uganda, Kenya, and Tanzania in eastern and southern Africa where rhodesiense HAT is endemic , 59, 62,se areas , 38, butStudying the human–livestock–wildlife interface is essential now, and even more so in the future because of human population expansion and agricultural development . This exIn this study, we identified both human- and animal-infective trypanosomes residing in cattle at the human–livestock–wildlife interface areas in Malawi. A limited number of trypanosomiasis and tsetse control programs addressing both diseases have been conducted in the area . AAT andT. b. rhodesiense, SRA PCR is still recommended. Trypanosome-susceptible cattle harbor both human and animal infective trypanosomes, implying its role as a potential reservoir of T. b. rhodesiense. This study emphasizes the urgent need for sustainable control measures within the context of the One Health approach for AAT and HAT in livestock.This is the first study to assess the prevalence of animal and human infective trypanosomes in cattle in Malawi. The use of MinION sequencing in combination with ITS1 PCR increased the reliability of PCR-based comprehensive trypanosome detection. However, for more sensitive and specific detection of human infective https://www.parasite-journal.org/10.1051/parasite/2020043/olmSupplementary material is available at Farmer registry list used during blood samples collection in the districts of Kasungu and Nkhotakota. (*) indicates the data obtained from the District Agriculture Development Office (DADO) for each district through their District Animal Health and Livestock Development Officer (DAHLDO).List of primers and their sequences used in this study.SRA PCR, ITS PCR, and MinION sequencing analysis for characterizations of trypanosome species. No hit* shows the number of reads that could not be identified by BLAST. FASTA $ describes the total number of obtained reads processed into BLAST. “PC+” and “?” refer to positive control and ambiguous results, respectively. Samples which were negative in both the SRA PCR and the ITS1 PCR are not shown here.Workflow for molecular detection of African trypanosomes. The workflow depicts structural steps for detection of African animal and human trypanosomes with serial arrows to the right for cattle samples, and a single downward arrow for human samples. The section on the identification of trypanosome species by sequencing is divided into three steps: library preparation, sequencing and basecalling, and de-multiplexing. Bioinformatic analyses required in the experiments are framed in red borderline.


Pichia kudriavzevii KKU20 and Candida tropicalis KKU20) was isolated from the rumen of dairy cattle. This study used 6 multiparous crossbreds between Holstein Frisian × Zebu dairy cows in their mid-lactation period. Dairy cows were randomly allocated to three ensiled RS with various yeast stains including Saccharomyces cerevisiae, P. kudriavzevii KKU20, and C. tropicalis KKU20 according to a 3 × 3 replicated Latin square design. Crabtree-negative yeast  increased the apparent digestibility of dry matter by about 6.9% when compared with Crabtree-positive yeast (S. cerevisiae). Bacterial populations were highest with ensiled RS by C. tropicalis KKU20. Ensiled RS with Crabtree-negative yeasts were significantly increased with total volatile fatty acids, but they did not affect volatile fatty acid profiles. Milk protein precentage was highest at 35.6 g/kg when C. tropicalis was fed, and lowest when applied with S. cerevisiae and P. kudriavzevii KKU20 in ensiled RS at 34.5 and 34.1 g/kg, respectively. Thus, feeding ensiled RS with novel Crabtree negative yeast could improve RS digestion, rumen fermentation, and milk protein content in dairy cows.Upgrading the nutritive value of rice straw (RS) is necessary to increase its contribution to enhancing meat and milk production. Present work verified whether novel Crabtree negative yeast inoculant could promote RS utilization, rumen fermentation, and milk quality in tropical crossbred lactating Holstein cows. The new stain of Crabtree negative yeasts ( Various approaches are applied to enhance the nutritional value and improve its utilization in dairy cow's nutrition2. The biological approaches are the most common with more harmless-treated RS4. For several years, yeast has become an innovative biological model organism for enhancing animal efficiency and is the traditional practice for ruminant feed additives5. Yeasts have been especially beneficial for single-cell protein creation and simply acceptable as their biomass has been used by ruminants in the form of fermented feed. In many studies, using yeast fermented with RS has been shown to enhance their nutritional value, silage quality, and nutrient digestibility6. S. cerevisiae, rapidly converts molasses and urea to provide biomass and greater nutrients from the whole-cell when added with oxygen (O2) during the proliferation process7. Wanapat et al.8 stated that S. cerevisiae significantly increases crude protein (CP) in feedstuff via cell proliferation during the fermentation process and it provides essential amino acids, particularly lysine and methionine, for dairy cattle. Previous studies explained the benefit of live yeast in that it could provide a positive effect on feed utilization and performance production in the ruminants10.As a result of rice production, million tons of rice straw (RS) are generated as a by-product; nevertheless, with low nutritional valuesS. cerevisiae has many benefits, several limitations have been reported, particularly that it produces low cell biomass6. Under aerobic conditions, S. cerevisiae exhibits alcoholic fermentation more than producing biomass. The “Crabtree-positive yeasts” are those that represent this characteristic11. Under excessive glucose and even aerobic conditions, Van Urk et al.12 revealed that S. cerevisiae had a limited proliferation capacity. Similarly, Wardrop et al.13 revealed that when cultivated with excessive glucose in a media solution, S. cerevisiae provides 7 times lower biomass compared to other strains. This phenomenon restricts the chances of animals to receive highly nutritious from yeast biomasses such as protein, essential amino acids, and vitamins. Consequently, it is important to extend the scope of research, and studying other yeast strains should be further improved. Crabtree-negative yeasts might be the most interesting option, as they have the special characteristic of limited fermentative products, biomass and carbon dioxide are the sole products under excessive glucose and aerobic conditions11. Unlike Crabtree positive yeast, excessive glucose did not inhibit affected on ethanol production. The pyruvate has used another channel for converted to cytosolic-acetyl coA via acetaldehyde and acetate through pyruvate dehydrogenase (PDH) bypass channel into mitochondria14. Consequently, high yeast biomass production more than Crabtree positive yeast may supply essential rumen fermentation factors and be a greatly nutritious feed supplement for ruminants.Although Pichia kudriavzevii KKU20 and Candida tropicalis KKU20) from rumen ensiled with RS and study their effect on feed digestion, ruminal fermentation, milk production, and milk composition of tropical crossbred lactating Holstein cows.Up to now, there is no research available information about the use of Crabtree negative yeast isolate from the rumen to improve RS. It was hypothesized that the novel Crabtree negative yeast inoculant could promote RS utilization, rumen fermentation, and milk quality in dairy cows. This research aimed to determine the effects of novel Crabtree-negative yeast , butyric acid (C4), and ammonia nitrogen (NH3-N) were ranged from 19.8 to 21.9, 5.1 to 5.4, 0.81 to 0.82, and 1.8 to 2.0 g/kg DM, respectively.According to the experiment of De Deken0.75. Organic matter (OM) and CP intake were 8.9 to 9.6 kg/day and 1.3 to 1.4 kg/day, respectively, which was not altered among treatments (P > 0.05). Crabtree-negative yeast  increased the apparent digestibility of DM by about 6.9% when compared with Crabtree-positive yeast (S. cerevisiae). However, the data achieved in this study showed that apparent digestibility of OM (OMD), CP (CPD), NDF (NDFD), and acid detergent fiber (ADFD) were not altered among yeast species and ranged from 762.1 to 791.5, 752.0 to 791.5, 601.5 to 641.3, and 492.8 to 525.4 g/kg, respectively. Furthermore, the total digestible nutrients were the same among yeast species and ranged from 734.8 to 767.7 g/kg (P > 0.05).The impacts of different yeast species ensiled RS on the effectiveness of feed utilization in dairy cattle is illustrated in Table 3-N, blood urea nitrogen (BUN), and microbial communities. Rumen pH was not changed among yeast species, and the pH values were 6.4 to 6.8 (P > 0.05). Ruminal NH3-N and BUN ranged from 16.5 to 22.1 mg/dL and 13.3 to 17.3 mg/dL, respectively (P > 0.05). The bacterial populations at both 0 h and 4 h after feeding and the mean value were highest (P < 0.05) with ensiled RS with C. tropicalis KKU20 by 9.9, 12.5, and 11.2 Log10 cell/mL, respectively. However, the fungal zoospore and protozoa populations were not affected by any treatments (P > 0.05).Table 2, C3, C4 proportions, and C2:C3 ratio are illustrated in Table P. kudriavzevii KKU20 and C. tropicalis KKU20 were significantly increased with a total VFAs at 0 h , and 4 h  after feeding (P < 0.05) when compared with S. cerevisiae, whereas yeasts ensiled RS had no effect on the VFAs’ profile (P > 0.05). The mean value of C2, C3, and C4 were 67.4, 22.3, and 10.3 mol/100 mol, respectively.The TVFA, CC. tropicalis KKU20 fed group at 35.6 g/kg (P < 0.01). Feed efficiency did not changed for any diets (P > 0.05).The effects of ensiled RS with various yeast species on milk production, composition of milk, and feed efficiency in dairy cows are shown in Table 15, CP and ME requirements of our animal  increased by about 1,190 g/day and 60.9 MJ/day, respectively. The nutrient composition in feed, especially CP  and ME (provide 79.05–84.14 MJ/day) values, were sufficient in our study for supporting dairy cows’ performance. Our study demonstrated that two yeasts were isolated from rumen fluids: P. kudriavzevii-KKU20 and C. tropicalis-KKU20. The name KKU refers to Khon Kaen University, where the strain was originally isolated, and the number “20” means the year of discovery, 2020. Presently, there are not many experiments has explored the impact of isolated yeasts on the mechanism of fermentation in the rumen. Intanoo et al.16 were isolated yeast from rumen fluids of three non-fistulated Thai-Holstein Friesian dairy cows and identified as Kluyveromyces marxianus and Pichia kudriavzevii for detoxifying aflatoxin B1 (AFB1) and it can apply further to use in animal feed. Correspondingly, Sirisan et al.17 found that there are three effective yeasts that could isolate from rumen fluids of Thai-Holstein Friesian dairy cattle including Pichia kudriavzevii, Candida rugosa, and Kodamaea ohmeri which are used as preventing acidosis in dairy cattle diets. Although, these yeast species had previously been isolated and used for the ruminant animal18. However, the qualities of these strains on ensiled RS and animal production have not been studied. This is the first time that yeast has been used in animal feed for performance testing.From NRCP. kudriavzevii KKU20 and C. tropicalis KKU20 (Crabtree-negative yeast) was established as having a low fiber content when compared with adding S. cerevisiae (Crabtree-positive yeast). The low fiber content can be clarified by the yeast’s ability to release cellulase enzymes and digest fiber during the fermentation process. Suntara et al.6 confirmed that C. tropicalis KKU20 and P. kudriavzevii KKU20 were more capable of releasing cellulase enzymes than S. cerevisiae by about 0.7 to 6.8 times, respectively. The experiment on in vitro gas production of ensiled RS at 14 days with the P. kudriavzevii KKU20 could decrease the NDF content by about 6.7% when compared with S. cerevisiae6. Ilmén et al.19 discovered yeast isolated from a plant named C. konsanensis species could excrete cellulase enzymes and digests fiber, and it is a new yeast strain that had not been reported previously. Similar to our study, C. tropicalis KKU20 and P. kudriavzevii KKU20 are great potential yeasts to improve feedstuffs and this study is the first report in ruminant nutrition feed research.Ensiled RS with 3-N level. Acceptable silage was defined by the pH value and the composition of their fermentation products20. The pH is the main indicator for evaluating silage quality and our study showed ensiled RS still has a satisfactory score of about 4.1 to 4.321. The pH is highly related to LA content, which in this study showed a consistent range of about 19.8 – 22.1 g/kg DM. LA content in silage should range between 21 to 25 g/kg DM to be considered of high quality, according to Flieg’s score22; This is close to the high quality of silage. Our result showed LA content similar to an earlier study by Suntara et al.6 who revealed that about 20.53 to 26.14 g/kg DM of LA was produced when ensiled RS with C. tropicalis KKU20 and P. kudriavzevii KKU20 at 14 days. NH3-N concentration in ensiled RS within the range of 1.80 to 2.00 g/kg DM indicated the normal standards for estimating silage. These results are similar to those of Li et al.23, who collected information on various types of RS parameters and concluded that RS silage has a NH3-N concentration of approximately 1.61 to 2.36 g/kg DM. Other parameters such as C2 show great value for preserved silage within the range of 20 to 25 g/kg DM21. After the fermentation process, the moisture content should range from 650 to 750 g/kg to be optimum24, which in our study showed an average of 722.1 g/kg. Hence, our study proposes that the nutrients in ensiled RS are still well preserved.The fermentation quality of ensiled RS with different yeast species indicated that the silage was well preserved. The ensiled RS still maintained appropriate pH, high lactic acid content, and a low NH25. This experiment indicated that Candida tropicalis is a Crabtree negative yeast. Radecka et al.26 stated that Pichia kudriavzevii shows ability as a crabtree-negative yeast species. Another feature that shows the difference between Crabtree-positive and negative, with oxygen as the final electron acceptor, respiration is possible under aerobic conditions, but Crabtree-positive yeasts (such as S. cerevisiae) would inhibit PDH and produce ethanol instead of respiratory complete resulted in low cell growth27. In contrast, “Crabtree-negative” mean yeasts neglect fermentative products, and biomass and carbon dioxide are the primary products under aerobic conditions12. According to Suntara et al.6 reported that the rate of growth was revealed to be lower in S. cerevisiae (Crabtree-positive yeast) than in Candida tropicalis and Pichia kudriavzevii (Crabtree-negative yeast) (P < 0.01). All of the above reasons were ensured that both yeast strains including Candida tropicalis KKU20 and Pichia kudriavzevii KKU20 used in this experiment were Crabtree negative yeast.The Crabtree effect can be measure from ratio, fermented glucose/ respiration glucose and positive if this ratio is > 1 and negative if the ratio is < 1. According to the experiment of De Deken28. Generally, the amount of RS that an animal intakes daily is limited to around 2.0% BW or less BW29. Because RS is rich in polysaccharides and has a high lignin and silica content, and thus it limits the voluntary intake30. However, Aquino et al.31 reported that the amount of RS that ruminants can consume can be as high as 1.2% BW, which is similar to our result of 0.8–1.0%BW. The intake of OM, EE, NDF, and ADF was similar to previous studies of lactating crossbred dairy cows33. The CP intake (CPI) in this study was also similar with Wanapat et al.2, which used lactating crossbred dairy cows (50% Holstein Frisian × 50% Thai native cows) and BW around 365.5 kg, and the CPI was about 1.0 to 1.2 kg/day. Typically, the CP found in tropical forage plants is often relatively low34. Especially in RS (%CP) when using a roughage source it can have an effect on the animal’s yield adequacy35. Our study showed that ensiled RS with yeast could support protein from yeast to low-quality roughage as RS, and the enhanced intake of protein was high enough to meet the requirement of tropical lactation dairy cows.Crabtree-negative or –positive yeast has no effect on the dry matter intake (DMI). Our results showed that the DMI (range from 2.6 to 2.8% BW) was similar to previous experiments, which is that feeding separate ensiled RS with a concentrate diet to dairy cows creates a DMI range from 2.5 to 3.2% BW6. The improved digestion may be due to the potential of how rumen microflora are promoted for better digestibility. Yeast is an important biological responder in rumen fermentation, live yeast cells improve microorganisms in rumen36 and stabilize pH in the rumen37. Habeeb38 stated that yeast could provide rumen with biological stimulants, which is necessary for microorganisms’ growth in the rumen and yeast contributes to establishing microbiota39 and is why the digestibility was apparently enhanced. This is consistent with Wang et al.9, who found that Crabtree-negative yeast as C. tropicalis could increase digestion in the in vitro technique and that it generated 3.03% more gas production than did S. cerevisiae.The DMD was increased when ensiled RS with Crabtree negative yeast was offered to animals. This strain is outstanding in terms of high proliferation ability and its high yield of cellulase enzymes6, who compared the effect of Crabtree-negative and -positive yeast on ensiled RS on the in vitro gas and confirmed that in the rumen, there was no difference among yeast species in NDFD and ADFD (705.2 vs 703.6 and 464.8 vs 464.4 g/kg DM).Crabtree-negative yeast did not change the apparent OMD, CPD, NDFD, and ADFD. The NDFD and ADFD are similar among Cabtree-negative and positive yeast . Noticeable changes occurred after the silage process was complete, but when the animal intakes the feed, its digestion was not altered. The reason for this is still not clear, but it is possible that yeast does not react directly on RS. Rather, digestion in the rumen occurred by the cooperation of microbes’ synergy until the resulting values were not statistically different. This is similar to an experiment by Suntara et al.P. kudriavzevii KKU20 and C. tropicalis KKU20 (Crabtree-negative yeast) could increase bacterial populations when compared to S. cerevisiae (Crabtree-positive yeast) by about 4.76%. The ruminal bacterial populations depend on sufficient nutrients or stimulants supply38. Yeast is a great supply to stimulate bacteria because it is enriched in essential substances40. Previous studies have confirmed that yeast could supply essential amino acids, vitamins, and minerals to increase the ruminal bacteria more than without yeast41. The key explanation is that under aerobic conditions, Crabtree-negative yeast may proliferate more than Crabtree-positive yeast since the enzyme mechanism functions differently42. Suntara et al.6 found that at 72 h of incubation time, P. kudriavzevii KKU20, C. tropicalis KKU20, and S. cerevisiae had growth by about 10.02, 9.6, and 8.87 Log cells/mL, respectively. The high amount of Crabtree-negative yeast creates a greater supply of essential nutrients to the rumen bacteria6, thus the amount of rumen bacteria is increased in response to the Crabtree-negative yeast.Ensiled RS with the 43. The great bacterial population could enhance carbohydrate digestion and then the animal obtains the greater VFAs44. This is similar to Castillo-González et al.45, who stated that the expansion of rumen microorganisms could increase the quantity of rumen VFAs. Certainly, a high bacterial population in our experiment was related to the Crabtree-negative yeast’s effect. Nonetheless, the direct influence of the Crabtree-negative yeast on rumen bacterial populations was unclear and this hypothesis required further research to be conducted. Expanding the Crabtree-negative yeast population (during the fermentation process) may be more effective than expanding that of the Crabtree-positive yeast (S. cerevisiae). This suggests that animals have a greater chance of obtaining stimulants for activating rumen bacteria. In agreement with our results, Wang et al.9 compared the effect between Crabtree-negative yeast  and Crabtree-positive yeast (S. cerevisiae) for in vitro gas technique and found that the inclusion of 0.25 × 107 of Crabtree-negative yeast could enhance the total VFAs by 7.7%. Suntara et al.6 reported that Crabtree-negative yeast (P. kudriavzevii KKU20) increased the total VFAs by 2.3% for in vitro gas study more than Crabtree-positive yeast.The ensiled RS with Crabtree-negative yeast has more effect on the total VFAs than with Crabtree-positive yeast by about 6.1% at the mean value. The high production of TVFAs in rumen fluids is related to the amount of ruminal bacteria46; 11.1 kg/h/day according to Wanapat et al.2. To produce milk, cows must calve and split their lactation cycle into four phases 47. The milk yield response was greater in the early lactation, and in the mid-lactation period, the milk yield begins to decline from its peak48. The lower actual milk yields in this study may be because dairy cows were in mid to late lactation (DIM 165.5 to 186.5). Our study indicated that daily protein yields in the milk of the C. tropicalis KKU20 group were highest at 35.6 g/kg and lowest when applied with S. cerevisiae and P. kudriavzevii KKU20 in ensiled RS at 34.5 and 34.1 g/kg, respectively. Milk protein is associated with the feed degradation energy supply as VFAs and microbial crude protein (MCP) synthesis49. High amounts of microorganisms in rumen could affect the MCP synthesis. This will be the supplied protein and amino acids (AA) in the small intestine and could enhance the milk protein yields50. Our result clearly demonstrated that C. tropicalis KKU20 was unique in the highest bacterial population (11.2 Log10 cell/mL), which is why the increase in milk protein yields occurred. Furthermore, there were no differences in milk proteins between S. cerevisiae and P. kudriavzevii KKU20. This suggests that the influence of Crabtree-negative yeast may play different roles in terms of milk quality. This thought is support by Intanoo et al.16, who compared different yeast strains that were in the same group of Crabtree-negative, and found that P. kudriavzevii KKU20 decreased daily protein yields in milk by 14.9% when compared with Kluyveromyces marxianus in crossbred lactating cows. This yeast species could provide high biomass, which possibly supplies more amino acid sources for milk protein synthesis. This is similar to Wardrop et al.13 who stated that K. marxianus has an outstanding ability to provide high biomass when compared with other strains. The explanation is limited in regard to P. kudriavzevii’s impact on daily protein yields in milk. A few studies have focused on applying non-S. cerevisiae to dairy cows and further research about the influence of each strain is required.The milk yield and milk composition of ensiled RS with Crabtree-negative yeast did not have any impact. Our study showed that the actual milk yields are about 8.5 to 8.8 kg/h/day, which are slightly lower than previous trials using early to mid-lactation cows , based on the Ethics of Animal Experimentation of the National Research Council of Thailand. Our study confirmed that all methods were performed in accordance with the relevant guidelines and regulations.17, the ruminal fluid was collected only once on day 7 from each fistulated steer via a rumen cannula at 4 h after the morning feed. Rumen fluids from two steers were mixed well before being filtered through cheesecloth folded to form 4 layers, then into a bottle, and were placed immediately into ice bucket (4 °C). They were then transported to the laboratory within 15 min. In the laboratory, for the total plate count, 1 mL of ruminal fluid from each animal was diluted with 0.85% sodium chloride to 1:10, 1:100, and 1:1000. Each ruminal fluid dilution was spread over a yeast–malt extract (YM) agar plate , which was then incubated at 39 °C for 72 h. The inoculant plate was dissolved in distilled water and sterilized for 15 min at 121 ͦC by autoclaving. The YM agar consisted of malt extract (3 g/L), yeast extract (3 g/L), peptone (5 g/L), agar (20 g/L), and glucose (10 g/L).Two fistulated–crossbred Holstein Friesian steers, averaging 350 ± 20 kg body weight, were used as rumen inocula donors. The animals were fed this diet to facilitate microorganism adaptation in the rumen, allowing this to occur for 7 days before the rumen fluid was obtained. According to Sirisan6 the isolated yeast has high potential to produce biomass and cellulase enzyme was selected to identification. DNA isolation from potential yeast was performed by boiling lysis buffer cells. The divergent D1/D2 domain of 26S rDNA was amplified with primers NL-1 (5′- GCA TAT CAA TAA GCG GAG GAA AAG-3′) and NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′). Amplification was performed in 100 µl of reaction mixture conditioning 100 ng of 2.5 U of Taq polymerase, genomic DNA, 40 mM of each primer, 20 mM of each dNTP, 1.5 mM MgCl2, and 10 mM Tris–HCl. The nucleotide sequences of the 26S rDNA D1/D2 domain were determined directly using PCR products, with slight modifications. Cycle sequencing of the D1/D2 domain was carried out with the forward primer NL1 (5′-GCA TAT CAA TAA GCG GAG GAA AAG-3′) and reverse primer, NL4 (5′-GGT CCG TGT TTC AAG ACG G-3′), using an ABI PrismTM BigDyeTM Terminator Cycle Sequence Ready Reaction Kit  according to the manufacturer’s instructions.According to Suntara et al.51. Dairy cows were randomly allocated to three ensiled RS with various yeast species including S. cerevisiae, P. kudriavzevii KKU20, and C. tropicalis KKU20 according to a 3 × 3 replicated Latin square design.This study used 6 multiparous crossbreds between Holstein Frisian × Zebu dairy cows in their mid-lactation period (165.5 ± 44.0 DIM) with an initial body weight of 363.9 ± 55.80 kg (average milk yield 8.58 kg/day) and a mean age of 5 years. The milk yield reported was slightly higher than the previous studies, in which Holstein Frisian × Zebu cow’s milk yields were 2,897 kg/year or 8.05 kg/day6. The P. kudriavzevii KKU20 and C. tropicalis KKU20 (considered as Crabtree negative yeast25) were tested for their high-potential on in vitro study, which has an outstanding benefit for feed digestion and in vitro gas production6. The S. cerevisiae (considered as Crabtree positive yeast25) was obtained from the commercial baker’s yeast . In Fig. 6 cells/mL) was multiplied in media solution including 250 g molasses  plus 10 g urea per 1000 mL of water. After that, the solution’s pH was then modified using formic acid  to reach a final pH of 3.552. Media solution directly into electromagnetic air compressor  flushed with oxygen to complete respiration for maximum cell growth at 72 h  and adjusted with a moisture content of 650–750 g/kg to provide sufficient ensilage conditions24. Fifteen kilograms of ensiled RS were put into plastic bags , and sealed with a vacuum machine . To ensure an anaerobic environment, the bags were securely sealed and fermented at room temperature for 14 days6.The ruminal yeasts were obtained by isolating, screening, and identifying the rumen of crossbred Thai-Holstein Friesian dairy cattle15. The ingredients and nutrient composition of ensiled RS and concentrate diet were provided in Table The feeding trial lasted for 63 days (21 days/period with 3 periods); dairy cows were held in independent pens and individually fed roughage and concentrate diets at 07:00 and 16:00. Ensiled RS offered ad libitum for all cows. The experimental diet was formulated by using the Khon Kaen Complete Feed (KCF) 2006 Program base on NRC53, NDF, ADF54, and acid-insoluble ash (AIA)55. Body weights were measured every period. The calculation of ME was based on the equation defined by Wachirapakorn et al.56:6.At the time of the feeding trial, orts were obtained and weights were collected every day, and the feeding rate was adjusted daily to yield orts between 50 to 100 g/kg of intake. Individual voluntary feed determined consumption difference between the feed offered and orts. Around 5 g/kg of the overall fresh fecal samples were split into two parts; the first part of each day for DM analysis and the second part were pooled at the end of each period. The pooled fecal samples (500 g) were stored at − 20 °C until analysis. At 60 °C, composite samples were dried, pressed through a steel filter of 1 mm for grinding , and then analyzed for DM (ID 967.03), ash (ID 492.05), ether extract , CP (ID 984.13) content58. The pH of the ensiled RS was measured by a pH meter using cold-water extracts  according to Pholsen et al.60. Silage fluid subsamples were centrifuged for 15 min at 6,000 rpm and the liquid above the solid residue was filtered using a 0.45-micron syringe filter. High-performance liquid chromatography (HPLC) devices  were used to conduct LA, C2, C3, and C4 analyses61. The NH3-N concentration was calculated according to the Kjeldahl process53. Jugular blood and rumen fluid samples were obtained at 0 and 4 h after feeding on the last day of each period. A blood sample (approximately 10 mL) was obtained in tubes containing 12 mg of ethylene diamine tetra-acetic acid (EDTA) from the jugular vein. The plasma was isolated by centrifugation for 10 min at 500×g and preserved at − 20 °C until BUN analysis, according to Abdallah et al.62. Approximately 200 mL of rumen fluid was collected from the rumen by a stomach tube connected to a vacuum pump. Rumen fluid was assessed immediately by the pH meter for determining the pH and temperature. Rumen fluid samples were then filtered through 4 cheesecloth layers. A fluid sample containing 5 mL of 1 mol/L of H2SO4 applied to 45 mL of rumen fluid was put into the bottle. The rumen fluid mixture was centrifuged for 15 min at 6000×g and used for analyzing the NH3-N53 and VFA 63. Ruminal bacteria, protozoa, and fungal zoospores were numbered under a hemocytometer using the direct counting method64.The 10 g of fresh silage was blended with 90 mL of sterilized water and stored at 4 °C65. Fat, protein, lactose, TS, and SNF concentrations were measured as weighted media depending on morning and afternoon milk yields per each test day by infrared methods using Milko-Scan 33.Yields of 4.0% FCM were calculated according to Rafferty et al.66:67:During the last 7 days of each experimental period, milk samples were taken according to the yield for morning and afternoon milking, preserved with 2-bromo-2 nitropropane-1, 3-dial, and stored at 4 °C until analysis by using Milko-Scan  for fat, true protein, lactose, total solids (TS), and solids-not-fat (SNF) content. Milk urea nitrogen (MUN) was estimated by the diacetyl monoxime method using UV/Vis-spectrophotometer  according to Aguilar et al.65.For each cow and period, feed conversion efficiencies were determined by dividing the average yield of actual milk and ECM by the respective DMI and digestible protein per yield of actual milk68 according to the model:Yijk, observation from cow j, receiving ensiled RS i, in period k; μ, the overall of mean, Sl, the effect of square ; Mi, effect of yeast species in RS silage ; Aj, the effect of cows ; Pk, the effect of period ; and εijk, the residual effect. Significant differences between individual means were evaluated using Duncan’s multiple comparison tests when a significant (P < 0.05) effect was detected69. Standard errors of means were calculated from the residual mean squares in the analysis of variance.All data from the experiment were analyzed according to a 3 × 3 replicated Latin square design using the GLM procedure


Alzheimer’s disease (AD) is the leading cause of dementia in the world, accounting for 50–75% of cases. Currently, there is limited treatment for AD. The current pharmacological therapy minimizes symptom progression but does not reverse brain damage. Studies focused on nonpharmacological treatment for AD have been developed to act on brain plasticity and minimize the neurotoxicity caused by the amyloid-beta (Aβ) peptide. Using a neurotoxicity model induced by Aβ in rats, the present study shows that physical (PE) and cognitive exercise (CE) reverse recognition memory deficits (with a prominent effect of long-term object recognition memory), decrease hippocampal lipid peroxidation, restore the acetylcholinesterase activity altered by Aβ neurotoxicity, and seems to reverse, at least partially, hippocampal tissue disorganization. According to the World Health Organization, Alzheimer’s disease (AD) is a global public health priority  peptides in the extracellular space, hyperphosphorylated tau protein, and brain oxidative stress are some of the pathological alterations found in AD . Cognitive training contributes to maintaining neural functions, promoting cognitive flexibility, decreasing oxidative stress, and improving the quality of life in patients. Evidence suggests that CE produces long-lasting improvements in the memory performance of older adults who experience a normal cognitive decline . Adult male Wistar rats were purchased from the Federal University of Santa Maria (RS/Brazil) and were housed at the institute’s vivarium under controlled temperature (23 ± 2°C) in a 12-h light-dark cycle, with food and water available n = 12/group): sham surgery (Control); Aβ-induced neurotoxicity model (Aβ); sham surgery and PE training (PE); sham surgery and CE training (CE); Aβ and PE training (Aβ + PE); and Aβ and CE training (Aβ + CE). After that, the animals were subjected to behavioral tests to evaluate recognition memory and to monitor control parameters. Finally, the rats were euthanized, and brain tissue was collected for biochemical testing (n = 8) or histological analyses , followed by 10 days of recovery from surgery and Aβ aggregation. After this period, animals were subdivided according to the treatments , resulting in six groups  was dissolved in saline solution  at a concentration of 100 μM and incubated at 37°C for 4 days to induce Aβ 25–35 aggregation initiation. A total volume of 2.0 μl of Aβ protein was injected into each hippocampus based on the Paxinos and Watson brain atlas coordinates  by stereotaxic microinjection using a Hamilton syringe and an infusion pump. After surgery, rats were returned to their home cages and were monitored for 10 days.Before PE training, the rats were habituated to a treadmill built for rodents  to avoid stress effects. The habituation was conducted for 3 days .After the rats were subjected to the “good runner protocol,” which consists of placing the animals on the treadmill without inclination for three consecutive days (velocity 8 m/min for 10 min), the level of trainability was evaluated with a score ranging from 1 to 5 points . In the end, the animals that maintained an average of three or more points were included in the exercise group.2) running test was performed to determine the individual intensity of exercise. For this, the rats started running at a low velocity, which was increased 5 m/min every 3 min until the rat was unable to keep running. The time to fatigue (min) and the work volume (m/min) were considered indirect measures of maximal indirect VO2  and elevated plus maze (PM) were used to analyze exploratory and locomotor activities and to evaluate anxiety state, respectively.In the OF, the rats were placed in the left quadrant of a 50 × 50 × 39 cm open field made with wood painted white, with a frontal glass wall. Black lines were drawn on the floor to divide it into 12 equal quadrants. Crossing and rearing, as measures of locomotor and exploratory activities, respectively, were measured over 5 min  and two enclosed arms (50 × 10 × 40 cm), with an open roof, arranged such that the two open arms were opposite to each other. The maze is elevated to a height of 50 cm. The total number of entries and the time spent in the four arms were recorded over a 5-min session  were evaluated, respectively  were quickly removed, and then the hippocampal tissues were immediately isolated from the brain and cleaned using ice-cold saline. Tissue samples were frozen in liquid nitrogen and stored at −80°C until biochemical analysis was performed.After euthanasia, the brain tissues of some animals  levels were measured by a spectrofluorometric method using 20,70-dichlorofluorescein diacetate  was added to 300 μl of working FRAP reagent in a microplate  assay. The working FRAP reagent was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeClAcetylcholinesterase (AChE) activity is a marker of the loss of cholinergic neurons in the forebrain. The AChE activity was assessed by the Ellman method  were anesthetized and transcardially perfused with phosphate-buffered saline (PBS) solution followed by 4% formaldehyde. Brains were removed, postfixed for 24 h in 4% formaldehyde, and cryopreserved in 30% sucrose overnight at 4°C. Then, the brains were frozen, and coronal brain sections (12 μm thickness) were cut in a Cryostat (LEICA CM3050S). The sections were stained by hematoxylin-eosin (HE), and a qualitative analysis of the morphological parameters were observed under an optical microscope (Olympus CX21).Some rats  were compared using ANOVA followed by Tukey’s The significance level was set at 0.05 for all variables.F = 1.171; P = 0.33; F = 3.279; P = 0.36; F = 0.7539; P = 0.58). These data are important since they guarantee that the results observed on memory tasks are related to procedures influencing learning and memory processes and not to other behavioral alterations.There were no differences between the groups in the number of rearings  = 2.11, P = 0.06; Aβ: t(11) = 1.08, P = 0.30; PE: t(11) = 0.70, P = 0.49; Aβ + PE: t(10) = 1.61, P = 0.14; CE: t(11) = 0.21, P = 0.83; Aβ + CE: t(11) = 1.42, P = 0.18; In the OR memory training session, the rats explored each object for a similar percentage of total exploration time  = 0.30, P = 0.0127; t(11) = 3.56, P = 0.0045; CE: t(11) = 4.18, P = 0.0015; t(11) = 1.32, P = 0.21; t(10) = 3.59, P = 0.0049; Aβ + CE: t(11) = 7.22, P = 0.001; In the STM test, the control animals spent more time exploring the novel object (Control: t(10) = 2.28, P = 0.04; PE: t(11) = 6.08, P = 0.0001; CE: t(11) = 4.54, P = 0.0008; LTM; t(11) = 0.74, P = 0.47; t(10) = 3.34, P = 0.0075; Aβ + CE: t(11) = 3.28, P = 0.007; Twenty-four hours after the training, rats in the Control, PE and CE groups explored the novel object for more than 50% of the total exploration time  = 0.85; P = 0.51; H(6) = 13.93; P = 0.016; U = 30, P = 0.026). The interventions overcome memory deficits induced by Aβ protein  and between Control and CE group .Considering the DI, used for comparison between the groups, no differences were found in STM test (t(10) = 3.43, P = 0.0064; PE: t(11) = 3.19, P = 0.0098; CE: t(11) = 3.77, P = 0.0031; t(11) = 1.97, P = 0.07; t(10) = 3.11, P = 0.011; Aβ + CE: t(11) = 2.29, P = 0.042; In the SR memory test session, Control, PE and CE rats explored the novel rat for a longer percentage of time than the familiar rat  = 0.822; P = 0.538; Considering the DI, used for comparison between the groups, no differences were found  = 2.767, P = 0.03; P = 0.033; We found differences between the groups in ROS levels, as measured by the DCFH test  = 4.464; P = 0.002; P = 0.004; P = 0.031 for Aβ vs. Aβ + PE; P = 0.012 for Aβ vs. Aβ + CE; Differences among the groups were found (H(6) = 16.44, P = 0.0057; P = 0.0144; Differences in total antioxidant capacity  were observed among the groups (H(6) = 18.70, P = 0.0022; P = 0.0080; P = 0.0321 for Aβ vs. Aβ + PE; P = 0.0092 for Aβ vs. Aβ + CE; The AChE activity was different between the groups  and decreased the total antioxidant capacity and acetylcholinesterase activity in the hippocampus, leading to STM and LTM recognition deficits. Also, in a previous study, our group performed the histopathological analysis of the cerebral cortex of the rats submitted to the same Aβ hippocampal infusion model and we observed that the cerebral cortex of these rats presented intense deposition of amyloid plaques (Martinez-Oliveria et al., It is important to consider that between the main regions where the Aβ deposits are in the AD patient brain are the hippocampus and cortex, structures that are associated with learning and memory function (Jing et al., The neuroprotective influence of PE on dementia is widely studied. Individuals who practice regular physical activity have a 30–40% reduced risk of AD development in comparison to physically inactive individuals (Aarsland et al., As impressively as PE training, cognitive training also promoted short and long-term learning in Aβ rats. There is clinically significant evidence to support the effectiveness of CE (Cui et al., It is important to highlight that both the physical and the cognitive training had a more prominent effect of LTM object recognition memory, since, for this type of memory, in addition to the ability to learn the task, it is noteworthy that the animals that received the Aβ infusion and underwent the different training had a significantly higher object DI than those that received beta-amyloid but did not perform any type of exercise. The same difference between the groups was not observed in STM OR. It is important to consider that STM and LTM consolidation involve different neurobiological mechanisms, as the increase of hippocampal protein and gene expression, that is observed in LTM consolidation but is not required for STM (Izquierdo et al., Neurodegeneration related to AD is most pronounced in hippocampal cholinergic neurons, which are directly related to cognitive function Gold, . In rat Many studies address protection and prevention strategies to avoid AD. The most effective type of strategy seems to act before the pathology. However, we also need to think about treatment for patients who already have the disease, and currently, the alternatives are few. Since the symptoms of AD start to appear approximately 20 years after the onset of pathophysiological hallmarks (Dubois et al., In summary, the model of intrahippocampal Aβ infusion used in this study caused hippocampal oxidative stress and damage, decreased antioxidant capacity, altered hippocampal tissue morphology, and promoted short- and long-term deficits in recognition memory that mimic those described or suggested to occur in AD. Surprisingly, both PE and CE induced memory consolidation in animals that received Aβ infusion, which normally presents deficits. The effect of PE and CE is more prominent in long-term object recognition memory. Still, PE and CE reversed the lipid peroxidation and acetylcholinergic activity alterations induced by hippocampal Aβ. Therefore, both PE and CE have the potential to be included in the treatment of AD, since both interventions could be used in humans.The datasets generated for this study are available on request to the corresponding author.The animal study was reviewed and approved by Animal Care and Use Committee from Federal University of Pampa—protocol n. 14/2017.LD, AG, CS, LL, B-HN, and DD performed the experiments, analyzed the data, and wrote the manuscript. PM-C was responsible for the conceptualization of the study and supervision, analyzed the data, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.