Immunization of sheep against Fasciola gigantica with glutathione S-transferase

Glutathione S-transferase in Fasciola gigantica (FgGST) was isolated by affinity chromatography, by which highly purified enzyme was obtained. FgGST on the SDS-PAGE showed three protein bands ranging 24.5-26.5kDa. Glutathione S-transferase (GST) activity was determined by HABIG method. FgGST was evaluated as vaccine alone or in combination with either aluminum hydroxide or saponin in sheep against F. gigantica infection. ELISA was used for detection of anti-FgGST IgG. After vaccination, all sheep were challenged with 120 metacercaria of F. gigantica. The results indicated that anti-GST IgG was not elevated after challenge. All sheep were slaughtered 24-26 weeks after challenge. The results indicated that, although after second vaccination, antibody titers rose markedly in GST-Al(OH)(3) and GST-saponin groups, but declined 4 weeks after challenge. No correlation between anti-GST IgG titers and protection was observed. The highest fluke burden reduction was observed in the group vaccinated with GST-saponin (32%), but this reduction was not statistically significant in comparison with the control group.     

Relation between erythrocyte reduced glutathione and glutamate concentrations in Korean Jindo dogs with erythrocytes possessing hereditary high activity of Na-K-ATPase and a high concentration of potassium

The concentrations of sodium, potassium, reduced glutathione (GSH) and free amino acids and Na-K-ATPase activity in erythrocytes were examined in 35 purebred Jindo dogs in Korea. The incidence of Jindo dogs with a high potassium concentration and high activity of Na-K-ATPase in erythrocytes (HK phenotype) was 25.7%. The erythrocyte GSH concentration in HK Jindo dogs varied widely, from 2.45 to 12.38 mmol/l of RBCs, and was positively correlated with the erythrocyte glutamate concentration. These results indicate that HK Jindo dogs have normal to very high levels of erythrocyte GSH, which might result from the varying quantity of Na-dependent glutamate influx in the erythrocytes.     

The levels of selenium and glutathione peroxidase activity in blood of sheep, cows and pigs.

The levels of selenium (Se) and glutathione peroxidase (GPX) in the blood of sheep, cows and pigs under farm conditions were examined. Sheep appear to form two distinct groups, namely high Se and GPX and low Se and GPX. The high group gave ranges of 133-249 ng/ml and 77-179 iu/g Hb for blood Se and GPX respectively, while the low group showed levels of 21-67 ng/ml and 2-20 iu/g Hb. Overall sheep blood showed a high correlation between Se and GPX (r = 0-92, P less than 0-001). Cow bloods formed one group, all having low Se and GPX levels except for a single outlier. Omitting this animal, the overall ranges were 9-72 ng/ml and 6-36 iu/g Hb for Se and GPX respectively. Blood Se and GPX activity were significantly correlated (r = 0-59, P less than 0-001). Pigs formed a single group also, with the difference that while their blood Se was high, the corresponding blood GPX activities were relatively low. Overall ranges were 93-193 ng/ml and 17-69 iu/g Hb for Se and GPX respectively. Correlation between blood Se level and GPX activity in this species was not significant (r = 0-27, P more than 0-1).     

Variations in sheep serum conglutinating and haemolytic activity for sheep erythrocytes sensitized by rabbit antibody

Based on conglutinating and haemolytic reactions with sheep erythrocytes (E) sensitized by rabbit antibody (A), three types of sheep sera were encountered. Type 1 sera do not conglutinate or haemolyse sheep E-rabbit A. Type 2 sera failed to conglutinate, but are haemolytically active. Type 3 sera have both activities. Serum from one type 1 sheep still failed to conglutinate 5 days after venepuncture but was now haemolytically active (i.e., type 2). Some sheep that initially had type 2 sera had, five days after an intraperitoneal injection of yeast cells, sera with conglutinating activity (type 3 sera). Type 1, 2 and 3 sera all had haemolytic activity with human E-sheep A indicator cells.Pooled type 3 sera have the highest conglutinating titres with sheep E-rabbit A after 10 min incubation at 39 °C. At this stage, the haemolytic titres were very low. From 10 min, the conglutinating titres decreased whereas the haemolytic titres gradually increased until 80 min. Optimal conglutinating activity required less rabbit A to sensitize sheep E than did haemolytic activity.     

The biological selenium status of livestock in Britain as indicated by sheep erythrocyte glutathione peroxidase activity.

The reliability of erythrocyte glutathione peroxidase activity as an indicator of selenium status in livestock is discussed. Based on this measurement, a survey is described of the biological selenium status of sheep on each of 329 farms in Britain. Results showed that 47 per cent of these farms were probably unable to provide grazing livestock with sufficient selenium to maintain blood levels greater than 0.075 microgram per ml. Increased selenium deficiency from the increasing use of home grown feeds as a major constituent of livestock rations may be causally related to the increase of white muscle disease and other selenium responsive diseases in Britain.     

Effect of acaricides on the activity of a Boophilus microplus glutathione S-transferase

In the present study, we report the effect of several acaricides on the enzyme activity of a Boophilus microplus recombinant glutathione S-transferase (rGST). GST was expressed in Escherichia coli and was purified with glutathione (GSH) affinity column chromatography. The kinetic constants were determined by reacting GST with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione. We report the effect of several acaricides on the enzyme activity of rGST. Some acaricides (ethion, amitraz, chlorpyrifos, DDT, cypermethrin, diazinon, ivermectin, deltamethrin and flumethrin) inhibited rGST. Contrarily, coumaphos had an activating effect. Although the accurate mechanisms of the B. microplus resistance to acaricides remain elusive, this work helps in understanding how acaricides can interact with GST.     

Total glutathione and glutathione reductase in bovine erythrocytes and liver biopsy

The goal of the present study was to measure the total glutathione level and glutathione reductase activity in bovine erythrocytes and liver biopsy. Five cows were injected intraperitoneally with DL-ethionine (12.5 mg/kg B.W.), and two control cows were injected with normal saline (0.9% NaCl). Ultrasonography guided liver biopsy, and blood samples were collected at 0, 4, 7 and 10 days after injection. The hepatic total glutathione level was significantly increased on Days 7 (p<0.05) and 10 (p<0.01), and hepatic glutathione reductase activity was significantly increased on Days 4 (p<0.05), 7 (p<0.01) and 10 (p<0.01). There were insignificant changes in the erythrocytic total glutathione level. The present study demonstrated that liver biopsy is a valuable tool for detecting oxidative stress and for diagnosing hepatic dysfunction in cattle from the viewpoint of the status of glutathione and glutathione reductase.     

Postnatal changes in the levels of 2,3-diaphosphoglycerate, reduced glutathione and some enzyme activities in the erythrocytes of lambs.

The concentration of 2,3-diphosphoglycerate, and the activities of the enzymes hexokinase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glutathione reductase and NADH-dependent methaemoglobin reductase in the erythrocytes of newborn and adult sheep were investigated. All the enzyme activities and the concentration of 2,3-diaphosphoglycerate were found to be significantly greater in the erythrocytes of newborn lambs than in those of adult sheep.     

The stability of glutathione peroxidase activity in plasma from cattle,pigs and sheep on storage in the presence and absence of glutathione

Ovine, bovine and porcine plasma glutathione peroxidase (GSH-Px) activity decreased on storage at both 4°C and 20°C. The ovine and bovine enzymes were significantly less stable than the porcine enzyme. The addition of GSH to a final concentration of 2 mmol/L to plasma samples at the commencement of storage retarded the loss of both ovine and bovine plasma GSH-Px activity. The ovine enzyme was unique in that after inactivation by storage at 4°C, incubation with GSH restored the enzyme activity. It is recommended that plasma GSH-Px should be assayed fresh, or otherwise stored at –20°C.     

The relationship between reproductive activity and blood calcium in the calcium-deficient hen.

1. The dependence of reproductive activity in the laying hen upon adequate calcium intake has been investigated. 2. The response of plasma luteinising hormone concentration in calcium-deficient, as compared with calcium-replete hens, to injections of luteinising hormone releasing hormone and progesterone suggested that the primary site of reproductive dysfunction is the hypothalamus rather than the pituitary gland. 3. Laying hens, when presented with a calcium-deficient diet, ceased to lay as the plasma ionised calcium concentration decreased to less than 1.0 mM, supporting the view that there is a threshold of blood calcium activity below which reproductive activity ceases. 4. Supplementation of the diet with calcium produced an immediate restoration of plasma ionised calcium concentration to normal, despite an interval of a few days before the plasma total calcium returned to normal and egg laying resumed.     

A possible relationship between haemonchosis and haemoglobin polymorphism in Merino sheep in Kenya.

It is known that sheep show polymorphism for haemoglobin in that three phenotypes (HbA, HbB AND HbAB) are normally found. Since it has been suggested, on the basis of haematological values, that HbA type sheep are more able to survive in Haemonchus contortus endemic environments, a preliminary study was conducted on the performance of set-stocked Merino ewes and lambs in relation to their haemoglobin phenotypes. The results provided some evidence that, in Merino sheep at least, those with HbA type show 'self-cure' more frequently and effectively than those with HbB and HbAB types. Examination of the PCV values and total body weights of Merino eres showed a similar trend, ie, the HbA types had the highest haematological parameters and the heaviest body weights, while the HbB types were consistently lowest. It would appear that the practical significance of these results justifies investigation on a larger scale.     

Erythrocyte selenium-75 uptake as a measure of selenium status in weaner sheep, and its relationship to erythrocyte glutathione peroxidase activity

The relationship between in vitro erythrocyte 75Se uptake (75Se uptake) and erythrocyte glutathione peroxidase (EGSHPx) activity was examined in weaner sheep during periods of selenium depletion and repletion, to determine whether 75Se uptake was better correlated than EGSHPx activity to the development of weaner nutritional myopathy. In the 2 trials conducted, only 3 of 45 Merino wether weaners developed clinical myopathy and histological lesions in skeletal muscles. The 75Se uptake values and EGSHPx activities in these 3 sheep were no different from those in the unaffected sheep. There was a significant negative correlation between 75Se uptake values and EGSHPx activities over the entire period of the trials. It could not be demonstrated that 75Se uptake was any better correlated than EGSHPx activity to the development of nutritional myopathy, and it was concluded that EGSHPx activity indicated selenium status better than 75Se uptake in weaner sheep.     

Expression and characterisation of a Psoroptes ovis glutathione S-transferase

The astigmatid mite Psoroptes ovis is the causative agent of sheep scab, a highly contagious parasitic disease of sheep. Infection causes severe allergic dermatitis, resulting in damage to the fleece and hide, loss of condition and occasional mortality. Interest in the P. ovis allergens led us to characterise a glutathione S-transferase (GST) which displays homology to GST allergens isolated from the house dust mite, Dermatophagoides pteronyssinus and the cockroach, Blatella germanica. A cDNA encoding a mu-class GST from P. ovis was expressed in Escherichia coli and the recombinant protein purified for biochemical analysis. SDS-PAGE analysis indicated that the purified product was homogeneous and had an apparent molecular weight of 30 kDa. The recombinant GST (rGST) is active towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB), whereas 1,2-dichloro-4-nitrobenzene (DCNB) is a poor substrate. The recombinant protein was also tested for recognition by IgE and IgG antibodies in serum from P. ovis na?ve and P. ovis infested sheep. Neither IgE nor IgG antibodies were detected to the rGST. Prausnitz--Küstner testing with rGST did not provoke a characteristic weal and flare response. Biopsies collected at the PK test sites were stained for eosinophils, neutrophils, mast cells and basophils. Neutrophil, mast cell and basophil counts were not significantly different to the controls. Eosinophil numbers were significantly higher than controls, but were not due to an IgE response.     

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.     

Quantification of plasma reduced glutathione, oxidized glutathione and plasma total glutathione in healthy cats

Glutathione is an important intracellular tripeptide with multiple functions. Abnormal glutathione metabolism is thought to play an important role in various diseases of cats. However, no data regarding concentration of plasma glutathione are available for domestic cats. This study discusses the development of a rapid, simple high pressure liquid chromatography method for measurement of reduced glutathione (GSH), oxidized glutathione (GSSH) and total glutathione (GSHt) in plasma, for the purpose of establishing baseline data for future studies. The mean concentrations of GSH, GSSH and GSHt were 4.51+/-1 microM; 19.44+/-3.79 microM (expressed as GSH equivalent) and 23.59+/-3.89 microM, respectively. This is the first report of plasma glutathione concentrations in this species.