Reliability of the glutaraldehyde test to measure gamma-globulin levels in foals and the use of this test to check colostrum intake of foals

The glutaraldehyde coagulation test is a semi-quantitative test used to determine the gammaglobulin concentration in serum. The purpose of this study was to examine the reliability of the different modifications of this test by determining the sensitivity, specificity, positive and negative predictive value, and the prevalence of hypogammaglobulinemia in foals. The results of the glutaraldehyde coagulation test were compared with the serum gammaglobulin concentration as a reference value, determined by measuring total serum protein and the serum protein spectrum. It was concluded that the glutaraldehyde coagulation test is a suitable test to use in the field for determining the serum gammaglobulin concentration in foals. The test has good sensitivity, specificity, positive and negative predictive value when using a 5% glutaraldehyde solution and when results are evaluated at 30 minutes for a serum concentration of 8 g/l and at 50 minutes for a serum concentration of 5.5 g/l, a concentration below which therapy is recommended.     

植物蛋白对干酪凝乳特性的影响

研究3种植物蛋白对干酪凝乳特性的影响,从而选出1种最佳的植物蛋白.从3种干酪的凝乳特性(凝乳时间、凝乳强度、乳清OD值)和干酪凝块的质构特性(弹性、黏聚性、咀嚼性)2方面进行对比分析,并对干酪凝块的感官品质进行模糊评判.结果表明:添加豌豆蛋白制得的干酪品质优于其他两种.     

Thromboelastography: a tool for measuring hypercoagulability, hypocoagulability, and fibrinolysis

Objective: To describe the technique of thromboelastography (TEG) and review the applications of this coagulation test in humans and small animals. Data sources: Data sources included scientific reviews and original research publications. Human data synthesis: TEG in humans has been used for documentation of hypercoagulable and hypocoagulable states and has been shown to be beneficial in patient management. Veterinary data synthesis: Clinical evaluation of TEG in veterinary medicine is limited; however, recent reports have documented evidence of hypercoagulability in dogs with parvovirus and protein‐losing nephropathy. Additionally, many of the research models may be relevant to veterinary patients. Conclusions: TEG provides information about coagulation that is not available through routine coagulation tests. The application of TEG monitoring to veterinary patients shows promise; however, prospective clinical studies are needed.     

基于蛋白组学体外分析益母草水煎液对补体和凝血级联通路相关活性因子的作用

旨在通过蛋白组学探索益母草水煎液对人真皮微血管内皮细胞(human dermal microvascular endothelial cells, HDMECs)凝血与抗凝血相关因子表达的调节作用。MTT法筛选益母草水煎液对HDMECs的安全浓度；使用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification, iTRAQ)技术分析50和100 μg·mL^-1益母草水煎液作用24 h后HDMECs蛋白表达谱的变化，通过对比各组蛋白谱的变化筛选出差异显著的相关通路，分析该通路中筛选到的可信差异表达蛋白(differentially expressed proteins, DEPs)，并选取可信DEPs中与凝血与抗凝血相关的拮抗因子进行RT-PCR和ELISA验证。结果表明：1 mg·mL^-1以下益母草水煎液对HDMECs无毒副作用；益母草水煎液能够同时调节与凝血相关的血小板活化等过程和与抗凝血相关的肝素结合过程。对筛选出的补体和凝血级联通路中的5种可信DEPs分析显示，与空白组相比，50 μg·mL^-1益母草水煎液组凝血酶原(F2)、抗凝血酶-Ⅲ(AT-Ⅲ)、组织型纤溶酶原激活剂(t-PA)、凝血因子Ⅴ(F5)和激肽原(KNG)同时显著下调，100 μg·mL^-1益母草组F2、t-PA、AT-Ⅲ、KNG显著下调；与50 μg·mL^-1益母草水煎液组相比，100 μg·mL^-1益母草水煎液组F2、AT-Ⅲ、t-PA、F5和KNG等凝血级联相关因子均显著升高。结果提示，益母草水煎液可显著改变HDMECs蛋白表达谱，并可能通过调控补体和凝血级联通路相关因子F2、AT-Ⅲ、t-PA、F5、KNG蛋白的表达对凝血与抗凝血相关拮抗因子发挥双向调控作用。     

木瓜蛋白酶凝固大豆蛋白质机理

本文研究了木瓜蛋白酶对豆浆的凝固机理，通过酶反应过程中游离氨基和蛋白质表面疏水性的变化，初步确定酶法凝固的机理是在酶的作用下，蛋白质暴露了一些疏水基,并立即通过蛋白质分子间的疏水相互作用结合成凝胶。     

New Models of Hemostasis

Hemostasis is an essential protective mechanism that depends on a delicate balance of procoagulant and anticoagulant processes. The waterfall/cascade models of coagulation are useful for understanding several essential steps of coagulation in vitro. These have resulted in the creation of the plasma-based tests used commonly and the ability to identify deficiencies in the extrinsic, intrinsic, and common pathways of coagulation. The model was also essential in elucidating the role of several of the inhibitors of coagulation and is currently used to demonstrate coagulation as it occurs in plasma in a static environment that is devoid of endothelial interactions. The intrinsic pathway originally described by these models does not appear to be essential for in vivo hemostasis but may play a role in pathologic thrombosis. The waterfall/cascade models' lack of cellular elements sets the stage for the cell-based model of coagulation. The cell-based model of blood coagulation, which includes the varied, complicated network of factors necessary for appropriate in vivo coagulation to occur, was the next step in the evolution of our understanding of coagulation. Recently, researchers have focused on real-time, in vivo models of hemostasis and this research reveals unexpected phenomena.     

Pharmacologic evaluation of factor XIIIa*-like enzyme activity in equine plasma as a potential therapeutic avenue for the inhibition of fibrinous tissue.

Several pharmaceutical compounds were evaluated for their ability to selectively inhibit activated coagulation factor-XIII-like enzyme activity (eg, XIIIa*) in pooled equine plasma. Presence of coagulation factor-XIIIa*-like enzyme activity in plasma was established by assay procedures involving incorporation of the fluorescent amine compound, monodansylcadaverine, into purified casein, which served as a protein substrate. Pharmaceuticals inhibitory to coagulation factor-XIIIa*-like enzyme activity were recognized by plasma gel formation of high spectrophotometric transmittance (transparency), solubility of transparent fibrin gels in concentrated urea solution, in conjunction with simultaneous depletion of native fibrinogen fractions, and production of fibrin monomer. Compounds acting primarily as anticoagulants were recognized by lack of plasma gel formation, but retaining high spectrophotometric transmittance and no detectable depletion of native fibrinogen fractions. Compounds failing to inhibit either thrombin-mediated fibrinogen-fibrin transformation (ie, coagulation) or coagulation factor-XIIIa*-like enzyme activity were recognized by opaque plasma gels caused by fibrin polymerization, low spectrophotometric transmittance values, and coinciding with depletion of native fibrinogen fractions. Pharmaceuticals capable of exerting selective inhibition of coagulation factor-XIIIa*-like enzyme activity were further classified as competitive inhibitors of phase 1 (carbamide) or phase 2 (terminal amine) of the transglutamination process.     

The relationship between factor XI coagulant and factor XI antigenic activity in cattle.

Factor XI protein, isolated from normal bovine plasma, was used to raise antiserum in rabbits. The antisera was partially purified and used in a neutralization-inhibition assay to investigate the relationship between factor XI coagulant activity and antigenic material in the plasma of normal cattle and cattle homozygous and heterozygous for factor XI deficiency. Factor XI antigen was reduced in both the homozygous and heterozygous animals to levels comparable to the factor XI coagulant activity. The reduction of immunologically cross-reactive material to normal factor XI suggests that the factor XI coagulation defect is associated with the absence of a normal protein.     

Relationship between assays of inflammation and coagulation: A novel interpretation of the canine activated clotting time

The processes of inflammation and coagulation are known to be interconnected through several mechanisms; however, the influence of inflammation on the interpretation of coagulation assays remains unknown. Blood was collected from 87 dogs admitted to a tertiary referral intensive care unit (ICU) and 15 control dogs. The association between 2 markers of inflammation [mature neutrophil count and C-reactive protein (CRP)] and 5 coagulation parameters [activated clotting time (ACT), prothrombin time (PT), activated partial thromboplastin time (aPTT), antithrombin (AT), and platelet count (plt)] were evaluated through correlation analysis. The study population was then divided into 4 groups based on severity of ACT prolongation with comparisons to all other variables assessed through an analysis of variance (ANOVA) test. A strong correlation for a biological system was demonstrated between ACT and CRP (r = 0.66; P < 0.0001). Statistically significant results were also found between aPTT and AT with the markers of inflammation, but the correlations were weaker. Within ACT groups of increasing severity, higher CRP concentrations (P < 0.0001) and lower AT activities (P < 0.0001) were identified. This study provides evidence for an association between assays of inflammation and coagulation and suggests that modification of our traditional interpretations of coagulation assays may be required. As a point-of-care test, ACT is a simple and inexpensive tool that can be used to assess an underlying inflammatory or hemostatic process.     

The effects of automated plasmapheresis on clinical, haematological, biochemical and coagulation variables in horses

The goal of this study was to determine the effects of plasmapheresis on the behaviour, general condition, haematological, biochemical and coagulation variables of donor horses for 32 days following the procedure. Twenty millilitres of plasma/kg body weight were collected via plasmapheresis in six clinically healthy horses. The general behaviour and condition of the horses was not affected by the procedure. During plasmapheresis, there was a mild increase in the haematocrit, haemoglobin concentration and total erythrocyte and leucocyte counts (P < 0.01). The mean concentrations of total protein and albumin decreased significantly (P < 0.01) and total protein did not normalise for about three weeks. Several other biochemical variables also decreased significantly during plasmapheresis, but mostly remained within reference ranges. After plasmapheresis, the mean value of the activated partial thromboplastin time and the thrombin time were mildly but significantly increased (P < 0.01), and the mean activities of factor V, factor VIII and antithrombin decreased significantly (P < 0.01), although all coagulation values remained within reference ranges. Our results indicate that, in horses, the collection of 20 mL of plasma/kg body weight via plasmapheresis results in mild changes in several haematological, biochemical and coagulation variables, although these were of no clinical relevance for the donors.     

Prekallikrein deficiency in a dog

A 15-year-old male dog with chronic hematuria was found to have prolonged activated partial thromboplastin time. Results of plasma coagulation assays indicated a deficiency of prekallikrein, a plasma protein that modulates the rate of activation of factors XI and XII in the intrinsic clotting system. Patients with prekallikrein deficiency rarely have manifestations of hemorrhage. The hematuria was the result of a renal transitional cell carcinoma.     

Comparison of in vitro tests for evaluation of passive transfer of immunoglobulins in giraffe (Giraffa camelopardalis).

Serum samples from captive giraffe (Giraffa camelopardalis) were tested to assess passive transfer of immunoglobulins using in vitro methods developed for domestic ruminants. Estimated immunoglobulin levels were compared using five tests (protein electrophoresis, total protein refractometry, zinc sulfate turbidity, glutaraldehyde coagulation, and sodium sulfite turbidity). A linear relationship was observed among total protein, gamma globulin (electrophoretic measurement), and immunoglobulin level based on spectrophotometric measurement of zinc sulfate turbidity. Nonquantitative assays also demonstrated statistical correlation with the quantitative methods. Using criteria similar to those established for domestic species, cutoff values for failure of passive transfer (FPT) were established for these tests in neonatal giraffe: 1) total protein <6.0 g/dl; 2) gamma globulin < 0.5 g/dl; 3) estimated immunoglobulin level < 1,000 mg/dl (zinc sulfate turbidity); 4) glutaraldehyde coagulation test negative; or 5) no visually detectable turbidity in 16% sodium sulfite or Bova-S negative. Retrospective examination of the medical histories showed a strong statistical association between animals designated as having FPT and those that were removed from their dams based on clinical assessment to be hand-reared. Application of these tests in the field should allow earlier detection and intervention for FPT in neonatal giraffe.     

三叶草叶蛋白提取工艺优化研究

作者选用三叶草为试验材料,通过正交试验L9(3⁴)分别对料水比、加盐比、不同pH和絮凝温度4个因素进行优化。以叶蛋白提取率、蛋白质量分数为指标,以期获得三叶草叶蛋白提取的最佳优化工艺参数。正交试验表明,提取三叶草叶蛋白的最佳提取工艺为A₃B₃C₁D₂,即料水比为1∶3、加盐量为5%、pH为3.0、絮凝温度为70 ℃为最佳的提取工艺组合。本研究初步用正交试验的方法对三叶草叶蛋白的最佳提取工艺进行了优化研究,为三叶草叶蛋白的开发应用提供了理论依据。     

Evaluation of a bench-top coagulation analyzer for measurement of prothrombin time, activated partial thromboplastin time, and fibrinogen concentrations in healthy dogs

OBJECTIVE: To evaluate a bench-top coagulation analyzer for determination of prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen concentration in healthy dogs. ANIMALS: 55 healthy adult dogs. PROCEDURES: PT, APTT, and fibrinogen concentration were determined by use of the coagulation analyzer. Values were compared with results obtained independently by a conventional laboratory. RESULTS: Correlations (with 95% confidence intervals) between the coagulation analyzer and conventional laboratory values were 0.760 (0.610 to 0.857), 0.700 (0.448 to 0.721), and 0.896 (0.878 to 0.918) for PT, APTT, and fibrinogen concentration, respectively. Using linear regression, comparison of data from the coagulation analyzer and the conventional laboratory provided equations relating the coagulation analyzer values with values from the conventional laboratory and suggested that APTT and fibrinogen values from the coagulation analyzer and conventional laboratory were approximately the same within expected random variation. Prothrombin time values for the coagulation analyzer were significantly offset from the PT values for the conventional laboratory but still were correlated reasonably well with the conventional laboratory values. CONCLUSIONS AND CLINICAL RELEVANCE: By use of the mechanical method of analysis, fibrinogen concentrations obtained with a bench-top coagulation analyzer correlated well with results for a conventional laboratory, indicating that the coagulation analyzer is a reliable instrument for determination of this coagulation variable. Coagulation analyzer results for PT and APTT correlated less strongly with those for the conventional laboratory, but they would still be considered clinically reliable.     

Proteomic analysis of plasma from Holstein cows testing positive for mycobacterium avium subsp. Paratuberculosis (MAP)

Johne's disease (JD) is a widespread and economically important chronic inflammatory disease of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Although there are several techniques available for diagnosis of JD, their sensitivity is questionable. New proteome profiling methods, such as serum/plasma protein fingerprinting by 2-Dimensional Fluorescence Difference Gel Electrophoresis (2D-DIGE), may therefore be useful for identifying novel protein biomarkers of MAP infection. In this study, plasma samples were collected from 380 Holstein cows and screened for the presence of MAP infection using the M.pt. Johne's antibody Kit (IDEXX). Five negative (MAP-), and 5 strongly positive (MAP+) cows were selected for proteomic analysis. Highly abundant proteins were depleted from the plasma samples using the ProteoMiner technology (Bio-Rad) to enhance the resolution of low abundance proteins. Plasma samples from MAP-, MAP+, and a pooled internal control were labelled with different fluorescent dyes and separated based on their isoelectrical point (IP) and then their molecular weight. Gel images of the fluorescent plasma protein maps were acquired using a Typhoon scanner and analyzed using the DeCyder software. Proteins that were differentially expressed were excised from the gels, trypsin digested, and subjected to MS/MS analysis for identification. Six proteins were identified as being up-regulated at least 2-fold in MAP+ cows including: transferrin, gelsolin isoforms α & β (actin binding protein - ABP), complement subcomponent C1r, complement component C3, amine oxidase - copper containing 3 (AOC3), and coagulation factor II (thrombin) (p<0.05). Two proteins that were down-regulated approximately 2-fold in the MAP+ cows included coagulation factor XIII -B polypeptide (COAFXIII), and fibrinogen γ chain (FGG) and its precursor.     

Soy Protein Isolate versus Meat-Based Low-Protein Diet for Dogs with Congenital Portosystemic Shunts

Background: Both presurgical preparation and long-term support of nonoperable dogs with congenital portosystemic shunts (CPSS) require optimal dietary management. Studies suggested that protein source may play an important role, with vegetable and dairy protein sources having better effects on hepatic encephalopathy (HE) than meat proteins.
Objectives: Determine whether a low-protein test diet with soy as its main protein source results in better scores than a control diet with the same composition but with poultry as its main protein source in dogs with CPSS.
Methods: In a double-blind cross-over study, 16 dogs received each diet for 4 weeks. Dogs in group T first received the test diet and then the control diet, whereas dogs in group C were fed the diets in the opposite order. Different variables (body weight, body condition score, HE score, fecal score, CBC, plasma tests of liver function including NH₃, and coagulation tests) were measured at the start of the study and after completion of each diet.
Results: One-way repeated measures ANOVA was performed. Plasma NH₃ was significantly lower after the test diet than after the control diet. The test diet also resulted in significantly higher fibrinogen concentrations and lower prothrombin times. The HE score improved with both diets, with no significant difference between the 2 diets.
Conclusions: Both diets achieved a significant improvement in HE score. The influence of the soy-based diet on plasma NH₃ concentration and coagulation parameters suggests that such a diet decreases the risk for HE and gives better support of liver function.     

Platelets enhance endotoxin-induced monocyte tissue factor (TF) activity in the horse

Endotoxaemia is the leading cause of death in horses. Disseminated intravascular coagulation (DIG), stimulated by induced monocyte proteins, is a prominent feature. Monocyte-platelet cellular interactions are central to the vascular dysfunction produced by circulating endotoxin and are implicated in many thrombotic diseases in the horse. This study reports that endotoxin (0.01-10 microg ml(-1)) and blood platelets (2.5 x 10(7) - 1 x 10(8) ml(-1)) are potent inducers of expression and activity of monocyte tissue factor (TF), the primary activator of the blood coagulation protease cascade.The co-incubation of endotoxin-stimulated monocytes with platelets resulted in greater production of this protein. Cycloheximide (1 mM) inhibited part of the stimulatory effect of endotoxin and/or platelets, the uninhibited part indicating de-encryption of cell-surface TF. Hence, platelets are considered to be an important component of the endotoxin-stimulated response of equine monocytes. The role of platelets as potent stimulators of endotoxin-stimulated monocyte proteins and mediators in vitro is likely to be of significance in vivo in the clinical manifestations and management of endotoxaemia in the horse.     

Coagulation defects of aflatoxin intoxicated rabbits

Twelve New Zealand white rabbits were intoxicated with aflatoxin B1. Most rabbits developed a coagulation defect near the time of death. Immediately prior to death there were significant decreases in factors V, VII, and VIII coagulant activities and fibrinogen concentration without a change in plasma fibrin(ogen) degradation product concentration, platelet number, and detectable plasma fibrin monomers. Microscopic evidence of disseminated intravascular coagulation was present in one rabbit with marked, diffuse hepatic necrosis. Terminal serum albumin concentration was significantly correlated to plasma factors V and VII activities and fibrinogen concentration. The coagulation defect of aflatoxicosis is primarily due to diminished hepatic synthesis of coagulation factors except when hepatic necrosis is severe enough to initiate intravascular coagulation and consumption of coagulation factors.     

Effects of total replacement of corn silage with sorghum silage on milk yield,composition, and quality

Background: In the last years, difficulties occurring in corn cultivation(i.e., groundwater shortages, mycotoxin contamination) have been forcing dairy farmers to consider alternative silages. Some experiments conducted on lactating cows have proven that the total replacement of corn silage with sorghum silage did not reduce milk yield.However, this kind of substitution involves supplementing sorghum-based diets with grains, to compensate for the lower starch content of sorghum silage compared to corn silage. Change of silage type and inclusion of starch sources in the diet would influence rumen fermentations, with possible effects on milk composition(i.e., fatty acid profile) and coagulation properties. A worsening of milk coagulation properties would have a negative economic impact in Italy, where most of the milk produced is processed into cheese.This study was designed to compare milk composition and quality, with emphasis on fatty acid profile and coagulation properties, in dairy cows fed two diets based on corn or sorghum silage.Results: The sorghum diet reduced milk yield(P = 0.043) but not 4% fat corrected milk(P = 0.85). Feeding sorghum silage did not influence milk contents of protein(P = 0.07) and lactose(P = 0.65), and increased fat content(P = 0.024).No differences emerged for milk concentrations of saturated(P = 0.61) and monounsaturated fatty acids(P = 0.50),whereas polyunsaturated fatty acids were lower(P 0.001) for the sorghum diet. Concentrations of n-6(P 0.001) and n-3 fatty acids(P = 0.017) were lower in milk of cows fed the sorghum diet. Milk coagulation properties did not differ between the two diets, except the "a30"(the curd firmness, expressed in mm, 30 min after rennet addition), that was lower(P = 0.042) for the sorghum diet.Conclusions: Feeding a forage sorghum silage, properly supplemented with corn meal, as total replacement of corn silage maintained milk composition and did not influence negatively milk coagulation properties, which have a great economic relevance for the Italian dairy industry. Thus, silages obtained from forage sorghums could have a potential as substitute of corn silages in dairy cow diets.