Capillary electrophoresis of the scrapie prion protein from sheep brain

Scrapie in sheep and goats causes a progressive, degenerative disease of the central nervous system and is the prototype of other transmissible spongiform encephalopathies (TSE) found in humans and in animals. In samples of TSE-affected brains, unique rod-shaped structures are found and are infectious. These rods are composed of a protease-resistant, post-translationally modified cellular protein (PrP^sc) that has a molecular mass of ca. 27 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Laboratory tests used for the diagnosis of scrapie detect PrP^sc. The overall concentration of PrP^sc in tissues is low. The present methods to diagnose scrapie are lengthy, require relatively large quantities of starting material to detect PrP^sc and lack sensitivity. We explored the use of free zone capillary electrophoresis and immunocomplex formation to detect PrP^sc in the brain tissue of infected sheep. Brain tissue from both infected (as confirmed by histological and biological tests) and from normal animals was used to prepare the PrP^sc. After treatment with proteinase K and non-ionic detergents, PrP^sc was solubilized and reacted with a rabbit antiserum specific for a peptide of the prion protein. Immunocomplex formation was observed for the samples from scrapie-infected brain but not for samples from normal brain. When a fluorescein-labeled goat anti-rabbit immunoglobulin was used as a second antibody, the detection of immunocomplex formation was enhanced both by the immunological technique and by using laser-induced fluorescence for detection. This same rabbit antiserum was used on immunoblot analysis. Three bands were observed for material from an infected sheep but none in preparations from brain material from normal sheep. Capillary electrophoresis can be used to show immunocomplex formation when PrP^sc is present in sheep brain.     

Intramolecular controls for the generation of clinical-quality SNP genotypes and the assessment of normal heterozygote imbalance

We present an inventive method that generates intramolecular controls for SNP analysis, termed Mirror SNPs. Using the ovine diseases of callipyge and scrapie as examples, we describe the PCR-driven production of balanced heterozygote copies of the various SNPs implicated in these diseases. In the absence of a callipyge-positive control DNA, we generated a balanced heterozygote Mirror SNP that represents both the wild-type and mutant forms of the causal polymorphism. Simultaneous analysis of this artificial Mirror SNP and the Real (target) SNP was used to prove the absence of the mutant form of the nucleotide at the Real SNP position in tested samples. Scrapie susceptibility was assessed using a PCR-driven system which generated four separate Mirror SNPs, and these enabled the confirmation of an apparent departure from 'balanced' heterozygote appearance at Real SNPs tested. Mirror SNP technology is generic and will enable the accurate assessment of rare and medically important SNP variants, more accurate frequency determinations, and the potential assessment of SNPs in 'mixed template' samples common in forensic analyses.     

Diagnostic value of different antigenic fractions of hydatid cyst fluid from camel and sheep in Kingdom of Saudi Arabia

Hydatid cyst fluids (HCF) crude extracts from camels and sheep slaughtered in Riyadh region, KSA were subjected to Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS–PAGE) and Western blot analysis. Sera from 17 confirmed human cases of hydatidosis, 25 patients with other parasitic infections and 10 clinically healthy subjects were used to evaluate the diagnostic value of the different antigenic fractions of these extracts. Immunoblotting results revealed that, at least 11 major discrete protein fractions (110–8 kDa) were recognized by sera from hydatidosis patients, sera from patients with other parasitic diseases showed cross-reactivity with few of these bands. The cluster of bands (38–35 kDa) that may be a breakdown of “Arc 5” antigen (39–38 kDa) was detected by 100% and 94% of sera from hydatidosis cases with HCF extracts from camel and sheep, respectively. This cluster showed also some cross reactivity (20% and 8%) with control sera from patients with other parasitic infections with camel and sheep HCF extracts, respectively. Polypeptides at 24–22, 16 and 8 kDa which may probably correspond to antigen B subunits were also identified by all samples from hydatidosis patients with sheep HCF extracts and by 100%, 65% and 74% with camel HCF extracts respectively. Sera from control subjects did not react with any of these polypeptides (24–22, 16 and 8 kDa). According to our results, the identified molecular weight bands (16 and 8 kDa using HCF crude extracts from sheep and 24–22 kDa using HCF crude extracts either from camel or sheep) represent good candidates for immunodiagnosis of hydatidosis.     

Use of capillary electrophoresis and fluorescent labeled peptides to detect the abnormal prion protein in the blood of animals that are infected with a transmissible spongiform encephalopathy.

Transmissible spongiform encephalopathies in humans and in animals are fatal neuro-degenerative diseases with long incubation times. The putative cause of these diseases is a normal host protein, the prion protein, that becomes altered. This abnormal prion protein is found mostly in the brains of infected individuals in later stages of the disease, but also can be found in lymphoid and other tissues in lower amounts. In order to eradicate this disease in animals, it is important to develop a system that can concentrate the abnormal prion protein and an assay that is very sensitive. The sensitivity that can be achieved with capillary electrophoresis makes it possible to detect the abnormal protein in blood. A peptide from the carboxyl terminal region, amino acid positions 218-232, was labeled with fluorescein during the synthesis of the peptide at the amino terminus. Antibodies that have been produced to this peptide were affinity purified and used in a capillary electrophoresis immunoassay. The amount of fluorescein labeled peptide in the capillary was 50 amol. Blood was obtained from normal sheep and elk, from sheep infected with scrapie and elk infected with chronic wasting disease. Buffy coats and plasma were prepared by a conventional method. After treatment with proteinase K, which destroys the normal protein but not the altered one, the blood fractions were extracted and tested in the capillary electrophoresis immunoassay for the abnormal prion protein. The abnormal prion protein was detected in fractions from blood from infected animals but not from normal animals. This assay makes a pre-clinical assay possible for these diseases and could be adapted to test for the abnormal prion protein in process materials that are used for manufacture of pharmaceuticals and products for human consumption.     

Solution structure of the sheep prion PrP〚142-166〛: a possible site for the conformational conversion of prion protein

In order to get deeper insight into the molecular forces responsible for prion pathogenic conversion, conformational properties of a synthetic linear peptide derived from the globular core of sheep prion protein were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. The studied peptide encompassing the 〚142–166〛 (in human numbering) region of sheep prion protein, folds in physiological conditions into a β-hairpin like tertiary structure, whereas, in the non-pathogenic form of protein and in trifuoroethanol (TFE), the region is engaged in largely α-helical conformation. Such structural duality of the fragment indicates a possible transconformational site within prion protein and may explain one of the early structural causes of prion diseases.     

Chemistry and Molecular Biology of Transmissible Spongiform Encephalopathies

Prion diseases are currently in the spotlight. Among them, the Creutzfeldt–Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy, or mad cow disease, are most commonly known. The term “spongiform” refers to the characteristic appearance of the lesions found in affected brains. It is likely that prion diseases originate from a causative agent that replicates independently of nucleic acids. Current research assumes that a structural isoform of prion protein, the scrapie form PrP^Sc, is the responsible pathogen. The three-dimensional structure, but not the amino acid sequence of the isoform differs from that of the normal cellular isoform, PrP^c. According to a widely accepted hypothesis, the normal isoform of the protein is converted by an autocatalytic process into the scrapie form upon contact with the latter. This hypothesis has not yet been proven. However, considerable progress has been made in the last few years, which might provide answers to many open questions about prion diseases, the subject of this review.     

Determination of abamectin and doramectin in sheep faeces using HPLC with fluorescence detection

An analytical procedure for the determination of abamectin and/or doramectin in sheep faeces has been developed. Avermectins were extracted from sheep faeces with acetonitrile, clean-up using solid phase extraction (SPE) and analysed by high performance liquid chromatography (HPLC) with fl uorescence detection after derivatization with N-methylimidazole.The method has a low detection limit (1.0 ng/g of moist sheep faeces), low quanti fi cation limit (2.5 ng/g of moist sheep faeces), good recovery in the range 66.4-80.8% for abamectin and 67.7-85.5% for doramectin as well as good repeatability (>85%). The method is applicable to the study of the time pro fi le of excretion in sheep faeces and also for ecotoxicological studies of both avermectins.     

Analytical procedure for determination of the time profile of eprinomectin excretion in sheep faeces

An analytical procedure has been introduced to enable study of the time profile of eprinomectin excretion in sheep faeces. Eprinomectin was extracted from sheep faeces with acetonitrile, the extract was cleaned by solid-phase extraction (SPE), and, after derivatization by reaction with N-methylimidazole, trifluoroacetic anhydride, and acetic acid, eprinomectin was analysed by high-performance liquid chromatography (HPLC) with fluorescence detection. The method has a low detection limit (1.0 ng g⁻¹ of moist sheep faeces), a low quantification limit (2.5 ng g⁻¹ of moist sheep faeces), good recovery (in the range 78.8 to 87.1%), and good reproducibility (RSD<10%). The method was used to study the time-profile of excretion of eprinomectin in sheep faeces after a single topical administration of 0.5 mg kg⁻¹ b.w. of the drug. Because of its good recovery, precision, and sensitivity, the method has also proved applicable to further ecotoxicological studies of eprinomectin. Figure Autochthonous Slovenian dairy breed sheep – Istrian Pramenka     

Development of a surface plasmon resonance-based assay for the detection of Corynebacterium pseudotuberculosis infection in sheep

Caseous lymphadenitis (CLA), a disease affecting sheep and goats, is caused by Corynebacterium pseudotuberculosis and is difficult to detect, especially at early stages in its development. A surface plasmon resonance-based biosensor assay for the detection of antibodies to the phospholipase D (PLD) exotoxin of C. pseudotuberculosis in sheep serum was successfully generated. It employed a recombinant form of PLD, which was immobilised, and all aspects of the assay including minimisation of non-specific binding, and the regeneration of the chip, were optimised. The applicability of the assay was initially demonstrated using sera collected from experimentally infected sheep and from sheep with no prior history of infection. The assay was then evaluated on a panel of clinical samples and the results obtained compared very favourably to those obtained by a double sandwich ELISA (over 90% similarity) and clearly verified its analytical value.     

Arsenic metabolism in seaweed-eating sheep from Northern Scotland

Cation exchange and anion exchange liquid chromatography were coupled to an ICP-MS and optimised for the separation of 13 different arsenic species in body fluids (arsenite, arsenate, dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TMA), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinoyl ethanol (DMAE) and four common dimethylarsinoylribosides (arsenosugars). The arsenic species were determined in seaweed extracts and in the urine and blood serum of seaweed-eating sheep from Northern Scotland. The sheep eat 2-4 kg of seaweed daily which is washed ashore on the most northern Island of Orkney. The urine, blood and wool of 20 North Ronaldsay sheep and kidney, liver and muscle from 11 sheep were sampled and analysed for their arsenic species. In addition five Dorset Finn sheep, which lived entirely on grass, were used as a control group. The sheep have a body burden of approximately 45-90 mg arsenic daily. Since the metabolism of arsenic species varies with the arsenite and arsenate being the most toxic, and organoarsenic compounds such as arsenobetaine the least toxic compounds, the determination of the arsenic species in the diet and their body fluids are important. The major arsenic species in their diet are arsenoribosides. The major metabolite excreted into urine and blood is DMAA (95 +/- 4.1%) with minor amounts of MMAA, riboside X, TMA and an unidentified species. The occurrence of MMAA is assumed to be a precursor of the exposure to inorganic arsenic, since demethylation of dimethylated or trimethylated organoarsenic compounds is not known (max. MMAA concentration 259 microg/L). The concentrations in the urine (3179 +/- 2667 microg/L) and blood (44 +/- 19 microg/kg) are at least two orders of magnitude higher than the level of arsenic in the urine of the control sheep or literature levels of blood for the unexposed sheep. The tissue samples (liver: 292 +/- 99 microg/kg, kidney: 565 +/- 193 microg/kg, muscle: 680 +/- 224 microg/kg) and wool samples (10470 +/- 5690 microg/kg) show elevated levels which are also 100 times higher than the levels for the unexposed sheep.     

Determination of Boron in Animal Materials by Reactor Neutron Induced Prompt Gamma-Ray Analysis

Boron concentration of plasma, feces, urine and body tissues from sheep fed with borated water (100 mg B/l) and tap water were determined by neutron-induced prompt gamma-ray analysis to elucidate boron metabolism. The B level in plasma and urine increased rapidly and the B content of feces increased greatly. The B concentrations in body tissues (liver, kidney, spleen, thyroid and muscle) of B dosed sheep were ten times higher than those of tap water administered sheep.     

Arsenic metabolism in seaweed-eating sheep from Northern Scotland

Cation exchange and anion exchange liquid chromatography were coupled to an ICP-MS and optimised for the separation of 13 different arsenic species in body fluids (arsenite, arsenate, dimethylarsinic acid (DMAA), monomethylarsonic acid (MMAA), trimethylarsine oxide (TMAO), tetramethylarsonium ion (TMA), arsenobetaine (AsB), arsenocholine (AsC), dimethylarsinoyl ethanol (DMAE) and four common dimethylarsinoylribosides (arsenosugars). The arsenic species were determined in seaweed extracts and in the urine and blood serum of seaweed-eating sheep from Northern Scotland. The sheep eat 2–4 kg of seaweed daily which is washed ashore on the most northern Island of Orkney. The urine, blood and wool of 20 North Ronaldsay sheep and kidney, liver and muscle from 11 sheep were sampled and analysed for their arsenic species. In addition five Dorset Finn sheep, which lived entirely on grass, were used as a control group. The sheep have a body burden of approximately 45–90 mg arsenic daily. Since the metabolism of arsenic species varies with the arsenite and arsenate being the most toxic, and organoarsenic compounds such as arsenobetaine the least toxic compounds, the determination of the arsenic species in the diet and their body fluids are important. The major arsenic species in their diet are arsenoribosides. The major metabolite excreted into urine and blood is DMAA (95 ± 4.1%) with minor amounts of MMAA, riboside X, TMA and an unidentified species. The occurrence of MMAA is assumed to be a precursor of the exposure to inorganic arsenic, since demethylation of dimethylated or trimethylated organoarsenic compounds is not known (max. MMAA concentration 259 μg/L). The concentrations in the urine (3179 ± 2667 μg/L) and blood (44 ± 19 μg/kg) are at least two orders of magnitude higher than the level of arsenic in the urine of the control sheep or literature levels of blood for the unexposed sheep. The tissue samples (liver: 292 ± 99 μg/kg, kidney: 565 ± 193 μg/kg, muscle: 680 ± 224 μg/kg) and wool samples (10 470 ± 5690 μg/kg) show elevated levels which are also 100 times higher than the levels for the unexposed sheep. Received: 29 February 2000 / Revised: 26 April 2000 / Accepted: 1 May 2000     

Determination of mineral balances in sheep offered feed with added cadmium and zinc

The optimum conditions for microwave digestion of herbage and faeces to determine mineral concentrations were obtained by varying sample mass, reagent and heating programme, and it was confirmed that the resulting element concentrations were the same as for certified reference material. The effects of feeding cadmium to sheep at a level that is typical of polluted regions (1 mg/kg) for ten days were investigated, as well as the possible amelioration of cadmium effects by adding 30 mg/kg Zn to the diet. Cadmium in the feed increased the cadmium balance and produced several mineral disturbances, in particular a reduction in sodium balance which is typical of renal tubular disorders. Including zinc in the diet as well as cadmium reduced the cadmium balance to a level similar to that of sheep that did not receive cadmium or zinc, which suggests that the zinc status is critical in determining whether cadmium in feed increases the cadmium balance in sheep.     

Evaluation of a preclinical blood test for scrapie in sheep using immunocapillary electrophoresis

An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 7-12 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.     

Determination of mineral balances in sheep offered feed with added cadmium and zinc

The optimum conditions for microwave digestion of herbage and faeces to determine mineral concentrations were obtained by varying sample mass, reagent and heating programme, and it was confirmed that the resulting element concentrations were the same as for certified reference material. The effects of feeding cadmium to sheep at a level that is typical of polluted regions (1 mg/kg) for ten days were investigated, as well as the possible amelioration of cadmium effects by adding 30 mg/kg Zn to the diet. Cadmium in the feed increased the cadmium balance and produced several mineral disturbances, in particular a reduction in sodium balance which is typical of renal tubular disorders. Including zinc in the diet as well as cadmium reduced the cadmium balance to a level similar to that of sheep that did not receive cadmium or zinc, which suggests that the zinc status is critical in determining whether cadmium in feed increases the cadmium balance in sheep. Received: 1 August 1997 / Revised: 8 December 1997 / Accepted: 10 December 1997     

Determination of triazine herbicides in sheep liver by microwave-assisted extraction and high performance liquid chromatography

A multi-residue method developed for the analysis of triazine herbicides, simazine, atrazine, propazine and prometryne, in sheep liver is presented. The method is based on microwave-assisted extraction (MAE) of sheep liver using methanol as extractant and analysis of extracts by high performance liquid chromatography (HPLC) and ultraviolet detection. MAE operational parameters, the solvent type and volume, extraction temperature and time, were optimized in detail with respect to extraction efficiency of the target compounds from sheep liver. The recoveries of the method at two different spiked levels were assessed by analyzing spiked liver samples and were found to be in the range from 90 to 102% with good precision (<11%).     

Nortestosterone: endogenous in urine of goats, sheep and mares?

For a number of species it is known that nortestosterone, either the alpha- or beta-epimer, can be of endogenous origin. For goats and mares similar results have not yet been published. As a follow-up on the experiments with cattle, a large number of urine samples per animal were collected from pregnant goats, sheep and mares. These samples were analysed for the presence of alpha- and beta-nortestosterone and alpha-estradiol using GC-MS. The results show that in the goats and mares studied alpha-nortestosterone is present during pregnancy. In this study no alpha-nortestosterone could be demonstrated in sheep. From our study and recently published data, however, it is proven that alpha-nortestosterone can occur endogenously.     

Variations in content and structure of glycosaminoglycans of the vitreous gel from different mammalian species

The vitreous of all species is composed of essentially the same type of extracellular matrix macromolecules organized to a transparent gel. In this study, the composition and fi ne chemical structure of the glycosaminoglycans (GAGs) in the vitreous gel from sheep and goat were determined and compared with those of human and pig vitreous gels. The results showed that, in all examined species; hyaluronan (HA) was the predominant GAG, whereas chondroitin sulphate (CS) was the minor one. In the vitreous gel of the most relative species, i.e. sheep and goat, higher amounts of both of HA and CS were estimated as compared with pig and human tissues. The distribution of hydrodynamic sizes of HA and CS was significantly differed among different species. All HA preparations consisted of molecules with great variability in hydrodynamic sizes. The relative proportions of the large HA molecules (size >1.8 x 10(6) kDa) were significantly higher in sheep and goat as compared with human and pig vitreous gel. The length of CS chains was also of larger size in sheep and goat (50 and 58 kDa, respectively) than the respective chains in human and pig vitreous gel (38 and 28 kDa, respectively). The sulphation patterns of CS preparations were determined following enzymic treatments, HPLC and capillary electrophoretic analyses. The human vitreous-derived CS chains showed quite different sulphation profile than that of CS isolated from other species, since 4-sulphated disaccharides were identified as the dominant moiety. In conclusion, significant compositional and structural variations between the vitreous matrixes of different species at the GAG level were identified. The functional significance of these species-dependent variations is discussed.     

Development and validation of a liquid chromatography and ion spray tandem mass spectrometry method for the quantification of artesunate, artemether and their major metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma

Recently, promising fasciocidal activities of artesunate and artemether were described in rats and sheep. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify artesunate, artemether and their metabolites dihydroartemisinin and dihydroartemisinin-glucuronide in sheep plasma. Protein precipitation with methanol was used for sample workup. Reversed-phase high-performance liquid chromatography (HPLC) was performed using an Atlantis C18 analytical column with a mobile phase gradient system of ammonium formate and acetonitrile. The analytes were detected by MS/MS using selected reaction monitoring (SRM) with electrospray ionisation in the positive mode (transition m/z 267.4 → 163.0). The analytical range for dihydroartemisinin, dihydroartemisinin-glucuronide and artesunate was 10-1000 ng/ml and for artemether 90-3000 ng/ml with a lower limit of quantification of 10 and 90 ng/ml, respectively. Inter- and intra-day accuracy and precision deviations were < 10%. Consistent relative recoveries (60-80%) were observed over the investigated calibration range for all analytes. All analytes were stable in the autosampler for at least 30 h (6 °C) and after three freeze and thaw cycles. The validation results demonstrated that the LC-MS/MS method is precise, accurate and selective and can be used for the determination of the artemisinins in sheep plasma. The method was applied successfully to determine the pharmacokinetic parameters of artesunate and its metabolites in plasma of intramuscularly treated sheep.