Molecular detection of Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos, Trypanosoma evansi and first evidence of Theileria sinensis-associated bovine anaemia in crossbred Kedah-Kelantan x Brahman cattle.

BACKGROUND: Serious disease outbreaks in cattle are usually associated with blood pathogens. This study aims to detect blood pathogens namely Theileria species, Anaplasma species, Candidatus Mycoplasma haemobos and Trypanosoma evansi, and determine their phylogenetic relationships and haemato-biochemical abnormalities in naturally infected cattle. METHODS: Molecular analysis was achieved by PCR amplification and sequencing of PCR amplicons of 18SrRNA gene of Theileria species, 16SrRNA genes of Anaplasma and Mycoplasma species, MPSP genes of T. orientalis and T. sinensis, MSP4 gene of A. marginale, 16SrRNA gene of Candidatus Mycoplasma haemobos, and RoTat1.2 VSG gene of Trypanosoma evansi, in sixty-one (61) clinically ill Kedah-Kelantan x Brahman cattle in Pahang, Malaysia. RESULTS: A total of 44 (72.13%) cattle were infected with more than one blood pathogen. Theileria species was the blood pathogen with the highest molecular detection rate (72.13, 95% CI 59.83-81.81%). Nucleotide blast analyses of all sequences demonstrated high degree of molecular similarity (98-100%) in comparison with their respective reference sequences. Analysis of 18SrRNA gene sequences of Theileria species and 16SrRNA gene sequences of Anaplasma species revealed Theileria sinensis and Anaplasma platys respectively as additional species detected in these cattle. MPSP-PCR analysis was conducted for further confirmation of T. sinensis. The blood picture of eight infected cattle groups revealed poikilocytosis, anisocytosis, rouleaux formation and degenerative left shift. High mean erythrocyte fragility values were common in infected cattle groups. Anaemia of the macrocytic normochromic type and spherocytes were observed in the T. evansi and Anaplasma platys + Theileria sinensis double species co-infected cattle group. Normocytic normochromic anaemia was observed in the T. sinensis infected cattle group. Significant (p < 0.05) increases in serum liver and kidney parameters, total protein, globulin, total and unconjugated bilirubin and decreased albumin values were observed in the T. evansi infected cattle when compared to clinically healthy cattle. CONCLUSION: We present the first evidence of Theileria sinensis-associated bovine anaemia (TSABA) in Malaysian cattle. Because of the high occurrence of bovine theileriosis and detection of A. platys, there is an urgent need for appropriate preventive and control measures against these blood pathogens.

In vitro effects of plant essential oils on non-specific immune parameters of red drum, Sciaenops ocellatus L.

Phytochemicals such as plant essential oils (EOs) have been reported to favour various activities in the innate immune system of fish. Thus, the aim of this study was to verify the in vitro effect of three different plant EOs (Ocimum americanum, Cymbopogon flexuosus and Melaleuca alternifolia) on non-specific immune parameters and erythrocyte osmotic fragility of red drum, Sciaenops ocellatus. Concentrations of each plant EO evaluated in preparations of head-kidney macrophages, blood leucocytes and blood plasma were as follows: 0.0 (control), 1.0, 2.0, 4.0, 8.0, and 16.0 µg/ml. Red drum head-kidney macrophages significantly increased extracellular superoxide anion production when exposed (20 h) to O. americanum EO (1.0-8.0 µg/ml) and C. flexuosus EO (2.0 and 4.0 µg/ml). The respiratory burst of blood leucocytes (NBT test) significantly increased in all concentrations when compared to the respective control group, for all EOs. At the highest concentration (16.0 µg/ml), C. flexuosus EO significantly inhibited the haemolytic activity of complement system in red drum blood after 1 h exposure. None of the tested concentrations significantly altered plasma lysozyme activity or erythrocyte osmotic fragility after exposing (1 h) red drum whole blood to each EO. This study demonstrated that these plant EOs are capable of triggering superoxide anion production in red drum leucocytes (head-kidney macrophages and/or blood leucocytes). In vivo studies are warranted to address their potential as immunostimulants in the diet of red drum and other aquacultured species.

Effect of 16 pure hydrocarbons on the stabilization and lysis of fish (mudskipper: Boleophthalmus dussumieri) erythrocytes.

The in vitro effects of polycyclic aromatic hydrocarbons (PAHs) on erythrocyte membrane stability of the mudskipper (i.e., Boleophthalmus dussumieri) were tested by using field concentrations, acute and chronic potency divisor concentrations. This was achieved by studying their lytic or antilytic effects on fish erythrocytes in critical hypotonic saline media. The interaction of PAHs acute potency divisor concentrations with mudskipper erythrocyte causes dramatic changes in the structure of the membrane. A significant difference (p<0.05) was found between the control and treatment groups of mudskipper erythrocyte exposed to acute potency divisor concentrations. No significant difference (p>0.05) was observed between the control and the treatment groups of mudskipper erythrocyte exposed to field concentrations. The results showed that chronic potency divisor concentrations of PAHs protect mudskipper erythrocyte against osmotic hemolysis. Our results could be extended to the use of Erythrocyte Osmotic Fragility (EOF) test as a biochemical marker of membrane toxicity in marine pollution biomonitoring. However, results showed that membrane stability is not an appropriate biomarker for PAHs pollution after short exposure duration.

Effect of myostatin gene mutation on erythrocyte osmotic fragility, hematological parameters and fatty acid composition of serum and erythrocyte membranes in piglets.

Fatty acid composition of serum and erythrocyte membrane, erythrocyte osmotic fragility and hematological parameters were estimated with the objective of determining effects of the gene mutation in one-week-old MSTN homozygous mutant (KO, MSTN-/-), heterozygous mutant (MSTN-/+) and wild type (WT, MSTN+/+) piglets (n = 4 each). Erythrocyte osmotic fragility, complete blood count (CBC), and fatty acid composition of serum and erythrocyte membrane were determined by flow cytometric analysis, automated hematology analyzer system, and liquid chromatography, respectively. Mean of median corpuscular fragility (MCF) was lower (P < 0.05, 0.001) in KO than MSTN-/+ and WT piglets. KO piglets had decreased (P < 0.05) white blood cell (WBC) count, lymphocyte (LYM) count, platelet (PLT) count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width-standard deviation (RDW-SD), red cell distribution width-coefficient volume (RDW-CV), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), platelet-large cell ratio (P-LCR), and an increased red blood cell (RBC) count when compared with MSTN-/+ and WT piglets. The ratios of unsaturated fatty acid (UFA) to saturated fatty acid (SFA) concentrations in serum and erythrocyte membranes of MSTN KO piglets were 2-fold and 4-fold higher compared to WT piglets (P < 0.001), respectively. In conclusion, MSTN KO piglets had a decreased erythrocyte osmotic fragility, and altered hematological profile and fatty acid composition of serum and erythrocyte membranes, as characteristic phenotype.

Supervised Cycling Training Improves Erythrocyte Rheology in Individuals With Peripheral Arterial Disease.

Peripheral arterial disease (PAD) results in insufficient flow to lower extremities. Aerobic exercise provides health benefits for individuals with PAD, but basic science behind it is still debated. Twenty-one PAD patients aged about 70 years with female/male as 7/14 were recruited. Among them, 11 were randomized to have supervised cycling training (SCT) and 10 to receive general healthcare (GHC) as controls. SCT participants completed 36 sessions of SCT at the first ventilation threshold within 12 weeks and the controls received GHC for 12 weeks. Ankle-brachial index (ABI), 6-min walk test (6MWT), peak oxygen consumption ( V ˙ O2peak), minute ventilation ( V ˙ E), minute carbon dioxide production ( V ˙ CO2), erythrocyte rheology, including the maximal elongation index (EImax) and shear stress at 50% of maximal elongation (SS1/2), and the Short Form-36 (SF-36) questionnaire for quality of life (QoL) were assessed before and 12 weeks after initial visit. SCT significantly decreased the SS1/2 as well as SS1/2 to EImax ratio (SS1/2/EImax) and increased the erythrocyte osmolality in the hypertonic region as well as the area under EI-osmolality curve. The supervised exercise-induced improvement of erythrocyte deformability could contribute to the increased peripheral tissue O2 delivery and was possibly related with increased V ˙ O2peak. The physiological benefit was associated with significantly increased ABI, 6-min walking distance, cardiorespiratory fitness, and SF-36 score. However, no significant changes in aerobic capacity and erythrocyte rheological properties were observed after 12-week of GHC. In conclusion, SCT improves aerobic capacity by enhancing erythrocyte membrane deformability and consequently promotes QoL in PAD patients.

Iron accumulation in the choroid plexus, ependymal cells and CNS parenchyma in a rat strain with low-grade haemolysis of fragile macrocytic red blood cells.

Iron accumulation in the CNS is associated with many neurological diseases via amplification of inflammation and neurodegeneration. However, experimental studies on iron overload are challenging, since rodents hardly accumulate brain iron in contrast to humans. Here, we studied LEWzizi rats, which present with elevated CNS iron loads, aiming to characterise choroid plexus, ependymal, CSF and CNS parenchymal iron loads in conjunction with altered blood iron parameters and, thus, signifying non-classical entry sites for iron into the CNS. Non-haem iron in formalin-fixed paraffin-embedded tissue was detected via DAB-enhanced Turnbull Blue stainings. CSF iron levels were determined via atomic absorption spectroscopy. Ferroportin and aquaporin-1 expression was visualised using immunohistochemistry. The analysis of red blood cell indices and serum/plasma parameters was based on automated measurements; the fragility of red blood cells was manually determined by the osmotic challenge. Compared with wild-type animals, LEWzizi rats showed strongly increased iron accumulation in choroid plexus epithelial cells as well as in ependymal cells of the ventricle lining. Concurrently, red blood cell macrocytosis, low-grade haemolysis and significant haemoglobin liberation from red blood cells were apparent in the peripheral blood of LEWzizi rats. Interestingly, elevated iron accumulation was also evident in kidney proximal tubules, which share similarities with the blood-CSF barrier. Our data underscore the importance of iron gateways into the CNS other than the classical route across microvessels in the CNS parenchyma. Our findings of pronounced choroid plexus iron overload in conjunction with peripheral iron overload and increased RBC fragility in LEWzizi rats may be seminal for future studies of human diseases, in which similar constellations are found.

Triglyceride-lowering effect of the aldose reductase inhibitor cemtirestat-another factor that may contribute to attenuation of symptoms of peripheral neuropathy in STZ-diabetic rats.

Hyperglycemia is considered a key risk factor for development of diabetic complications including neuropathy. There is strong scientific evidence showing a primary role of aldose reductase, the first enzyme of the polyol pathway, in the cascade of metabolic imbalances responsible for the detrimental effects of hyperglycemia. Aldose reductase is thus considered a significant drug target. We investigated the effects of cemtirestat, a novel aldose reductase inhibitor, in the streptozotocin-induced rat model of uncontrolled type 1 diabetes in a 4-month experiment. Markedly increased sorbitol levels were recorded in the erythrocytes and the sciatic nerve of diabetic animals. Osmotic fragility of red blood cells was increased in diabetic animals. Indices of thermal hypoalgesia were significantly increased in diabetic rats. Tactile allodynia, recorded in diabetic animals in the early stages, turned to mechanical hypoalgesia by the end of the experiment. Treatment of diabetic animals with cemtirestat (i) reduced plasma triglycerides and TBAR levels; (ii) did not affect the values of HbA1c and body weights; (iii) reversed erythrocyte sorbitol accumulation to near control values, while sorbitol in the sciatic nerve was not affected; (iv) ameliorated indices of the erythrocyte osmotic fragility; and (v) attenuated the symptoms of peripheral neuropathy more significantly in the middle of the experiment than at the end of the treatment. Taking into account the lipid metabolism as an interesting molecular target for prevention or treatment of diabetic peripheral neuropathy, the triglyceride-lowering effect of cemtirestat should be considered in future studies. The most feasible mechanisms of triglyceride-lowering action of cemtirestat were suggested.

Effect of fisetin and probiotic supplementation on erythrocyte osmotic fragility, malondialdehyde concentration and superoxide dismutase activity in broiler chickens exposed to heat stress.

The aim of the study was to evaluate effects of fisetin and probiotic on erythrocyte osmotic fragility (EOF), malondialdehyde (MDA) and superoxide dismutase (SOD) in broiler chickens exposed to heat stress. Sixty day-old broilers were divided into: Group I (control) given distilled water; Group II, fisetin (5 mg/kg); Group III, probiotic Saccharomyces cerevisiae (4.125 × 106 cfu/100 ml); and Group IV, fisetin (5 mg/kg) + probiotic (4.125 × 106 cfu/100 ml) orally for 7 days. Blood samples collected from 42-day-old birds were evaluated for EOF, serum MDA concentration and SOD activity. Percentage EOF at 0.5% NaCl was lower (P<0.05) in fisetin, probiotic and fisetin + probiotic groups (34.26 ± 0.98%, 35.65 ± 0.81% and 34.25 ± 1.98%, respectively) than in controls (48.42 ± 0.40%). The MDA concentrations in broiler chickens administered with fisetin (14.37 ± 1.15 nmol/l), probiotic (5.66 ± 1.06 nmol/l) and fisetin + probiotic (4.136 ± 0.58 nmol/l) were lower (P<0.05) than in controls (22.64 ± 2.95 nmol/l). Activities of SOD were higher (P<0.05) in fisetin, probiotic and fisetin + probiotic broiler chickens (6.34 ± 0.24 IU/l, 5.67 ± 0.09 IU/l and 5.93 ± 0.13 IU/l, respectively) than in controls (5.37 ± 0.09 IU/l). Fisetin + probiotic ameliorated oxidative stress changes in broiler chickens better than fisetin or probiotic alone. In conclusion, administration of fisetin or probiotic and, especially their combination, decreased EOF, lipoperoxidation and increased superoxide dismutase activity in broiler chickens exposed to heat stress.

Cryohemolysis, erythrocyte osmotic fragility, and supplementary hematimetric indices in the diagnosis of hereditary spherocytosis.

BACKGROUND: Hereditary spherocytosis (HS) is a chronic hemolytic anemia characterized by microspherocytes in the peripheral blood and increased erythrocyte osmotic fragility (EOF). This study evaluated the cryohemolysis test (CHT); initial hemolysis (IH); immediate and incubated hemolysis percentage in 5.5 g/L NaCl (H5.5); mean corpuscular hemoglobin concentration (MCHC); red blood cell distribution width (RDW); and Hb/MCHC, Hb/RDW, and MCHC/RDW ratios for the diagnosis of HS. METHODS: Data from 13 patients with HS were evaluated at the Instituto de Bioquímica Aplicada and compared with data from 14 unaffected individuals and 11 patients with anemia due to another etiology. Total blood and reticulocyte counts, CHT, and immediate and incubated EOF were performed in all subjects; sensitivity, specificity, efficiency, and Youden index (YI) were calculated. RESULTS: Eight patients with HS had MCHC ≥345 g/L, 10 had RDW ≥14.5%, 12 had IH >5.0 g/L, 11 had immediate H5.5 ≥5%, and 13 had incubated H5.5 ≥50% (the cut-off value to consider HS). The efficiency and YI were: immediate H5.5 (0.94-0.85), incubated H5.5 (0.89-0.82), IH (0.89-0.78), MCHC (0.87-0.62), CHT (0.84-0.54), and Hb/MCHC (0.71-0.56), respectively. The calculated ratios could distinguish subjects with HS from unaffected individuals (P<0.05), but not those with anemia of another etiology (P>0.05). CONCLUSION: Although the CHT and supplementary hematimetric indexes were useful to differentiate individuals with SH from healthy controls, they cannot distinguish from anemias of other etiology. CHT and MCHC, in addition to EOF, are recommended for diagnosing HS patients because of their low cost and efficiency.

In vivo ameliorative effects of methanol leaf extract of Lawsonia inermis Linn on experimental Trypanosoma congolense infection in Wistar rats.

The aim of this study was to investigate the ameliorative effect of Lawsonia inermis Linn used traditionally against trypanosomosis. Twenty-five adult Wistar rats of both sex were individually infected intraperitoneally (IP) with 106Trypanosoma congolense per ml of blood. Following establishment of infection, the rats were randomly divided into five groups of 5 rats each. Rats in groups I, II, and III were treated with 125, 250 and 500 mg/kg of the extract, respectively, while rats in groups IV and V were treated with 3.5 mg/kg and 2 ml/kg of diminazene aceturate (DM) once and physiological buffered saline, respectively. All treatments except DM were given orally for 7 days IP. The antitrypanosomal effect of the plant was assessed by observing the level of parasitaemia daily, packed cell volume (PCV) weekly, erythrocyte osmotic fragility (EOF) and malondialdehyde (MDA) concentration on day 21. Phytochemical screening of the extract revealed the presence of alkaloids, carbohydrates, triterpenes, steroids, cardiac glycosides, saponins, tannins and flavonoids. The extract significantly (P < 0.05) reduced levels of parasitaemia at 250 mg/kg. PCV was higher (P > 0.05) in extract treated groups but significantly higher (P < 0.05) in group II at week 2 when compared to group V. Rats in group II had significantly lower values of EOF and MDA when compared with groups IV and V. Thus, the leaf of L. inermis has in addition to an antitrypanosomal effect against T. congolense in rats, an attenuating effect on the trypanosomosis pathology probably mediated via protection of the erythrocyte membrane against trypanosome-induced oxidative damage to the erythrocytes.

Flavonoid mixture ameliorates increase in erythrocyte osmotic fragility and malondialdehyde concentration induced by Trypanosoma brucei brucei-infection in Wistar rats.

The experiment was performed with the aim of investigating the effect of a flavonoid mixture, Daflon® 500 mg (DF) on the erythrocyte fragility and lipoperoxidative changes, induced by Trypanosoma brucei brucei infection in Wistar rats. Fifty adult male rats randomly divided into five groups of 10 animals each were used. Rats in the control group were administered (1 mL/kg) distilled water only, while the other groups were infected with T. brucei brucei and treated with Daflon® 500 mg and/or Diminazene aceturate. At the end of 5 weeks, EDTA-blood samples and serum samples were collected from the rats, and were used to determine erythrocyte osmotic fragility (EOF) and serum malondialdehyde (MDA) concentration respectively. The results showed that EOF and MDA concentration significantly (P<0.05) increased in the infected untreated group when compared to the treatment groups. Treatment with Daflon® 500 mg and Diminazene aceturate significantly (P<0.05) reduced trypanosome-induced increases in EOF and lipoperoxidative changes, suggesting possible antioxidant properties of Daflon® 500 mg and its therapeutic value in trypanosomosis.

Chlorpyrifos induced toxicity in reproductive organs of female Wistar rats.

Chlorpyrifos (CPF) is an organophosphate (OP) insecticide extensively used in agricultural and domestic settings. Healthy adult female albino rats were divided into three groups of six rats in each. Two groups were dosed orally with CPF in vegetable oil (0.1 and 2.5 mg/kg/day) and third group was given vegetable oil for 8 weeks. Non-significant changes were observed for body weight and feed intake. A disruption in estrous cyclicity was observed with a prolonged metestrous. Erythrocyte osmotic fragility and lipid peroxidation levels increased significantly. Mammary gland whole mounts revealed a significant (P<0.05-0.0001) increase in the ductal thickness, number of branches, alveolar and terminal end bud number and terminal end bud diameter. A significant increase in ovarian surface epithelium height, follicular diameter and follicular atresia was observed in treated rats (P<0.05-0.0001). A similar significant increase in the uterine surface epithelium height, endometrial gland epithelium height and myometrium thickness in higher dose group was recorded (P<0.05-0.0001). Luminal epithelium height and endometrial gland diameter was increased significantly in both the treated groups (P<0.05-0.0001). The results indicate that sub-chronic exposure of CPF causes oxidative stress and negative effects on the reproductive organs of female rats, which may be a pointer towards beginning of cancer incidence.

Biomedical studies on lipid peroxidation and erythrocyte fragility during the process of aging.

OBJECTIVE: To investigate oxidative stress, hemoglobin percentage and erythrocyte osmotic fragility in various aging groups. METHODS: A total of 200 healthy volunteers of both genders between age group 20-65 years were selected by random method. Determination of hemoglobin percentage was done employing modified cyanide method of Dacie and Lewis. The erythrocyte lysis was observed in hypotonic solution of buffered saline at varying concentrations and optical density was measured at 540 nm. The extent of lipid peroxidation in form of malondialdehyde was measured by thiobarbituric acid method. RESULTS: The study found a significant decrease in hemoglobin percentage, increase in erythrocyte osmotic fragility and increased lipid peroxidation in form of malondialdehyde with increasing age. CONCLUSIONS: Supplementation of antioxidants may prevent the oxidative injury in elderly group of subjects.

Variaciones de la Fragilidad Osmótica Eritrocitaria en Bovinos a Pastoreo sobre praderas con bajo contenido de Selinio y Suplementados o no con Selenio / Variations of the Erythrocyte Osmotic Fragility in Cattle Grazing on Pastures with Low Selenium Content with or Without Supplement with Selenium

El selenio es constituyente de la enzima antioxidante glutatión peroxidasa (GSH-Px; E.C.1.11.1.9). La deficiencia de selenio en bovinos disminuye la actividad sanguínea de GSH-Px, predisponiendo a las membranas celulares a sufrir un daño oxidativo. La susceptibilidad de los eritrocitos bovinos al estrés oxidativo puede estimarse mediante la prueba de fragilidad osmótica eritrocitaria (FOE). Con el propósito de evaluar la FOE en bovinos a pastoreo sobre praderas con bajo contenido en selenio, en diferentes periodos del año, se emplearon dos grupos de 16 vaquillas, que pastoreaban praderas con un contenido de selenio inferior a 0,04 ppm. En el mes de marzo, un grupo (Se+) fue suplementado con selenio (Deposel®) y el otro grupo (Se-) se mantuvo sin suplementación. Muestras de sangre fueron obtenidas de cada vaquilla previo a la suplementación y luego mensualmente en 10 ocasiones. La FOE se determinó utilizando la técnica descrita por Schalm. La actividad de GSH-Px se midió usando un reactivo comercial (Ransel®). La suplementación con selenio incrementó la actividad de GSH-Px a valores considerados adecuados (> 130 U/g Hb). En el grupo Se-, la actividad de GSH-Px fluctuó entre 60-100 U/g Hb. La FOE no registró diferencias entre grupos a la concentración de NaCl 0,55%; sin embargo, ambos grupos mostraron una disminución de la FOE, es decir un aumento en la resistencia de las membranas, desde septiembre, coincidente con el periodo de primavera en el hemisferio sur. Al parecer la pradera en este periodo favorece el aporte de nutrientes antioxidantes relevantes en la protección de las membranas.

Selenium is a part of antioxidant enzyme glutathione peroxidase (GSH-Px; E.C.1.11.1.9). In cattle, the selenium deficiency has been associated with decreased activity of glutathione peroxidase leading to an increase in oxidative damage of cells. The oxidative stress in erythrocytes from cows can be estimated by erythrocyte osmotic fragility test (EOF). To assess EOF as biomarker of oxidative stress in cattle grazing on pastures with low selenium content during different seasons of the year, two groups of sixteen heifers each were used. They were grazed on pastures with 0.04 ppm selenium content. In March one group (Se+) was supplemented with selenium (Deposel®) and another (Se-) was maintained unsupplemented. Blood samples were obtained from each heifer prior to supplementation and then one time each month for ten months. The EOF was determined using a technique described by Schalm. The GSH-Px activity was measured using a commercial kit (Ransel®). The GSH-Px activity was increased by selenium supplementation to adequate levels (> 130 U/g Hb). The GSH-Px activity in Se- group was 60-100 U/g Hb. No differences between groups were detected in the EOF values, at 0.55% NaCl concentration; however, both groups showed decreased EOF values after September, coincident with the spring season in the southern hemisphere. Seemingly in this period, the more rapidly growing pasture grass increased in levels of some others factors that lead to increased protection against oxidative damage to erythrocytes.