iTRAQ-based quantitative proteomics discovering potential serum biomarkers in locoweed poisoned rabbits.

Locoism threatens the sustainable development of animal husbandry in areas around the world with intensified desertification, especially in the western United States, western China, Canada, and Mexico, among other countries. This study was conducted to discover potential serum biomarkers in locoweed-poisoned rabbits and lay a foundation for early diagnosis of locoism. We performed iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry (2D LC-MS/MS), comparing locoweed-poisoned rabbits and healthy controls. A total of 78 differentially-expressed proteins (fold change > 1.5 or < 0.67) were identified in the locoweed-poisoned rabbits compared to healthy controls. We found that 57.70% of differentially-expressed proteins were functionally related, and through bioinformatics analysis, we were able to construct a network mainly in complement and coagulation cascades. Significant differences in thrombospondin 4 (THBS4), kininogen 1 (KNG1), hemoglobin (HBB), and complement factor I (CFI) between locoweed poisoned animals and controls were found (P < 0.05) and validated by western blotting. These results suggested that locoweed could damage neurocytes, lower immunity, and form thrombi in rabbits. Our study proposes potential biomarkers for locoism diagnosis and also provides a new experimental basis to understand the pathogenesis of locoism.

Molecular characterization of Capra hircus lysosomal α-mannosidase and potential mutant site for the therapy of locoweed poisoning.

Lysosomal α-Mannosidase (LAM) belongs to the glycoside hydrolyzing enzymes family 38 and is involved in the biosynthesis and turnover of N-linked glycoproteins process. Locoweeds, which contain swainsonine (SW) that inhibits LAM, are the main poisoning plants in many regions of the world, and thereby resulting in animal poisoning or even death. Based on regions of protein sequence conservation between LAM from Bos taurus and Homo sapiens, we cloned cDNA encoding Capra hircus LAM (chLAM). Expression of cDNA in Pichia pastoris resulted in the secretion of aLAM activity into the culture medium. The recombinant chLAM was activated 1.6 and 1.2-fold with Zn(2+) and Ca(2+), respectively. By homology modeling, molecular docking and mutant analysis, we obtained the probable binding modes of SW at the allosteric sites of chLAM, and the potential mutant sites for the resistance to SW. Prediction of SW sensitivity to A28 W/G, D58 Y/G mutant chLAM is lower than wild type chLAM. The obtained results lead to a better understanding of not only interactions between substrate/SW and chLAM, but also of a potential strategy for a novel therapy for locoweed poisoning.

The biogeochemistry of selenium in Sunan grassland, Gansu, Northwest China, casts doubt on the belief that Marco Polo reported selenosis for the first time in history.

In order to clarify the historic academic problem of whether or not livestock poisoning in ancient Suzhou of Northwest China, recorded by Marco Polo in 1295, was selenosis, this study deals with the biogeochemistry of selenium in Sunan County in the Hexi Corridor, which is part of ancient Suzhou in China. It was found that quite a number of farm animals had suffered from intoxication and died as a result of grazing poisonous grasses, mostly Oxytropis DC, Stellera chamaejasme, and Achnatheru inebrians. Toxic symptoms of livestock grazing on Oxytropis DC are similar to those of selenium toxicity, for instance, hair loss and hoof lesions as described by Marco Polo. Therefore, we thought that toxic grass, probably Oxytropis DC, led to the intoxication of livestock recorded by Marco Polo. Average Se concentrations in two members of this species were 0.112 +/- 0.038 mg/kg for the root of Oxytropis glabra, 0.102 +/- 0.027 mg/kg for the stem and leaf of Oxytropis glabra, and 0.066 +/- 0.009 mg/kg for Oxytropis ochrocephala. The average soil selenium concentration was 0.205 +/- 0.127 mg/kg on grassland producing Oxytropis glabra and 0.152 +/- 0.024 mg/kg on grassland producing Oxytropis ochrocephala. The average Se concentration in other plants was 0.076 mg/kg in the root of Ephedra monosperma Mey, 0.029 mg/kg in the root of Rheum palmatum, 0.031 mg/kg in the root of Stellera chamaejasme, 0.037 mg/kg in Achnatherum inebrians, and 0.067 mg/kg in forage grass (Achnatherum splendens ohwi). Selenium concentrations in soils and plants in Sunan County are far less than the thresholds causing selenium toxicity in livestock. As a result, this study concludes that the livestock poisoning recorded by Marco Polo in 1295 might not have been selenosis.

Effect of lonophore supplementation on selected serum constituents of sheep consuming locoweed.

The effects of ionophore supplementation on selected serum constituents of sheep consuming locoweed were investigated. Sixteen sheep were allotted by weight to a 2x2 factorial arrangement of treatments: 1) no locoweed, no lasalocid, 2) no locoweed, 0.75 mg lasalocid/kg BW, 3) 0.5 mg swainsonine/kg BW, no lasalocid, 4) 0.5 mg swainsonine/kg BW, 0.75 mg lasalocid/kg BW. Swainsonine was provided by locoweed (Oxytropissericea), and sheep were fed a blue grama based diet at 2.5% BW for a 35 d treatment period. Diets were formulated to be isocaloric and isonitrogenous. Blood samples were collected on d 1, 7,14, 21, 31 and 35 to determine serum swainsonine concentration, alkaline phosphatase, total iron, aspartate aminotransferase, g-glutamyltransferase, and lactate dehydrogenase activity and total cholesterol, and triglyceride concentrations. No lasalocid by locoweed interaction (P > 0.4) was noted for any response variable measured. Average daily gains (P = 0.4) and orts (P = 0.7) were not affected by the treatments. No lasalocid treatment (P = 0.7) or day (P = 0.1) effect of serum swainsonine was observed. A locoweed by day interaction (P < 0.0001) of serum alkaline phosphatase was detected. Alkaline phosphatase levels were elevated (P < 0.01) for locoweed treated sheep at 24 h following initial exposure and remained elevated throughout the trail. Total iron was suppressed (P < 0.08) in locoweed fed sheep. A day effect (P < 0.02) was observed for serum iron. However, no linear, quadratic, or cubic effects of day were noted (P >0.2). A locoweed by day interaction (P < 0.0001) of serum aspartate aminotransferase and g-glutamyltransferase was detected. Aspartate aminotransferase levels were elevated (P < 0.0001) by d 7 for locoweed treated animals and remained elevated throughout the trial. g--Glutamyltransferase levels were suppressed (P < 0.0001) by day 7 for locoweed treated animals and remained suppressed throughout the trial. A locoweed by day interaction (P = 0.06) of serum cholesterol was detected. However, no linear, quadratic, or cubic effects of day were detected (P = 0.2). Lasalocid treatment had no effect on any serum constituent measured. Use of lasalocid in grazing animals should not increase the likelihood of locoweed intoxication.

Dose response of sheep poisoned with locoweed (Oxytropis sericea).

Locoweed poisoning occurs when livestock consume swainsonine-containing Astragalus and Oxytropis species over several weeks. Although the clinical and histologic changes of poisoning have been described, the dose or duration of swainsonine ingestion that results in significant or irreversible damage is not known. The purpose of this research was to document the swainsonine doses that produce clinical intoxication and histologic lesions. Twenty-one mixed-breed wethers were dosed by gavage with ground Oxytropis sericea to obtain swainsonine doses of 0.0, 0.05, 0.1, 0.2, 0.4, 0.8, and 1.0 mg/kg/day for 30 days. Sheep receiving > or = 0.2 mg/kg gained less weight than controls. After 16 days, animals receiving > or = 0.4 mg/kg were depressed, reluctant to move, and did not eat their feed rations. All treatment groups had serum biochemical changes, including depressed alpha-mannosidase, increased aspartate aminotransferase and alkaline phosphatase, as well as sporadic changes in lactate dehydrogenase, sodium, chloride, magnesium, albumin, and osmolarity. Typical locoweed-induced cellular vacuolation was seen in the following tissues and swainsonine doses: exocrine pancreas at > or = 0.05 mg/kg; proximal convoluted renal and thyroid follicular epithelium at > or = 0.1 mg/kg; Purkinje's cells, Kupffer's cells, splenic and lymph node macrophages, and transitional epithelium of the urinary bladder at > or = 0.2 mg/kg; neurons of the basal ganglia, mesencephalon, and metencephalon at > or = 0.4 mg/kg; and cerebellar neurons and glia at > or = 0.8 mg/kg. Histologic lesions were generally found when tissue swainsonine concentrations were approximately 150 ng/g. Both the clinical and histologic lesions, especially cerebellar lesions are suggestive of neurologic dysfunction even at low daily swainsonine doses of 0.2 mg/kg, suggesting that prolonged locoweed exposure, even at low doses, results in significant production losses as well as histologic and functional damage.