Effect of zinc supplementation on ecto-adenosine deaminase activity in lambs infected by Haemonchus contortus: highlights on acute phase of disease.

Haemonchus contortus (order Strongylida) is a common parasitic nematode infecting small ruminants and causing significant economic losses worldwide. It induces innate and adaptive immune responses, which are essential for the clearance of this nematode from the host. Ecto-adenosine deaminase (E-ADA) is an enzyme that plays an important role in the immune system, while Zinc (Zn) has been found playing a critical role in E-ADA catalysis. Therefore, the aim of this study was to assess the effect of Zn supplementation on E-ADA activity in serum of lambs experimentally infected with H.contortus. To reach this purpose 28 male lambs (in average 25 kg) were used. The animals were divided into four groups: A and B composed of healthy animals (uninfected); C and D, infected with H.contortus. Groups B and D were supplemented with Zn Edetate, subcutaneously with 3 mg kg of live weight, on days 11 and 25 post-infection (PI). Blood and fecal samples were collected on the days 11, 25 and 39 PI, in order to assess hematocrit, seric E-ADA, and eggs per gram (EPG) counting, respectively. The animals of groups C and D showed severe hematocrit reduction (days 25 and 39 PI) and were EPG positive (days 11, 25 and 39 PI). On day 41 PI, three animals each group were subjected to necropsy. This procedure showed that animals of groups A and B did not have helminths in abomasum and intestines, while H.contortus were observed in groups C (5782.5 ± 810.9) and D (6185.0 ± 150.0). Infected and untreated animals (group C) showed a reduction in E-ADA activity, but this was not observed when the animals were supplemented with Zn (Group D). Therefore, based on our results, it was possible to observe that Zn supplementation exercised a positive effect on E-ADA activity in lambs infected with H.contortus, and did not allow a reduction in E-ADA activity, as occurred in the group infected and without supplementation. However, Zn supplementation was not able to prevent the worm burden.

Immunoreactivity of protein tyrosine phosphatase A (PtpA) in sera from sheep infected with Mycobacterium avium subspecies paratuberculosis.

Evasion of host defense mechanisms and survival inside infected host macrophages are features of pathogenic mycobacteria including Mycobacterium avium subspecies paratuberculosis, the causative agent of Johne's disease in ruminants. Protein tyrosine phosphatase A (PtpA) has been identified as a secreted protein critical for survival of mycobacteria within infected macrophages. The host may mount an immune response to such secreted proteins. In this study, the humoral immune response to purified recombinant M. avium subsp. paratuberculosis PtpA was investigated using sera from a cohort of sheep infected with M. avium subsp. paratuberculosis and compared with uninfected healthy controls. A significantly higher level of reactivity to PtpA was observed in sera collected from M. avium subspecies paratuberculosis infected sheep when compared to those from uninfected healthy controls. PtpA could be a potential candidate antigen for detection of humoral immune responses in sheep infected with M. avium subspecies paratuberculosis.

E-ADA activity in erythrocytes of lambs experimentally infected with Haemonchus contortus and its possible functional correlations with anemia.

The aim of this study was to evaluate the ecto-adenosine deaminase (E-ADA) activity in erythrocytes of lambs experimentally infected with Haemonchus contortus, correlating it with the degrees of anemia of the experimental animals. A total of 14 healthy lambs, with negative fecal exam for parasites, were to carry out the present study. They were divided into two groups, composed by seven animals: Group A represented the healthy animals (uninfected), while in Group B the animals were infected with 15,000 larvae of H. contortus. Blood was drawn on the days 15, 45 and 75 post-infection (PI) in order to perform the hematological analysis, as well as the mensuration of E-ADA activity in erythrocytes. Parasitological stool exam were performed on the same days mentioned above to follow up the evolution of the infection, as well to determine the number of eggs per gram of feces (EPG). On day 15PI, the animals presented negative EPG and there was not significant (P>0.05) difference between groups in relation to E-ADA activity and hematologic parameters. Animals in Group B had positive EPG for helminths on days 45 and 75 PI, accompanied by varying degrees of anemia, when compared to Group A. At the same periods E-ADA activity was significantly (P<0.05) increased in the erythrocytes of animals of Group B when compared with the not-infected ones. Statistically, there was a negative correlation (P<0.01) between activity E-ADA in erythrocytes and hematocrit on days 45 (r = -0.76) and 75 (r = -0.85)PI. Based on these results and in the scientific literature, it is possible to conclude that the E-ADA may participate on mechanisms related with the pathogenesis and host response against anemia caused by H. contortus.

Periacinar liver fibrosis caused by Tephrosia cinerea in sheep.

Tephrosia cinerea has been associated with ascites and liver fibrosis in sheep in Brazil. The dried plant was fed ad libitum to three sheep for 55-80 days. Three additional sheep were used as controls. All the treated sheep presented with hypoalbuminemia and increased γ-glutamyltransferase and aspartate aminotransferase activities. Anorexia, apathy, rough coat, ascites, and emaciation were observed after 45-60 days of feeding with T. cinerea. At necropsy 55-80 days after feeding of the plant commenced, the treated sheep had ascites, hydrothorax and hydropericardium, and their livers were firm and whitish, with a nodular surface. Histologically, the main hepatic lesions were periacinar fibrosis associated with hemorrhages and necrosis. On electron microscopy, a severe swelling of sinusoidal endothelial cells, frequently obstructing the lumen of the sinusoid was observed. The space of Disse was compressed by the swollen endothelial cells and microvilli usually present on the surface of hepatocytes adjacent to the space of Disse were not apparent. Dense bundles of collagen fibers were present in the spaces of Disse and within the sinusoids between profiles of swollen endothelial cells. It is concluded that T. cinerea causes periacinar fibrosis, similar to poisoning by Galenia africana in sheep and goats and veno-occlusive disease in different species.

E-ADA activity in serum of lambs experimentally infected with Haemonchus contortus.

The aim of this study was to evaluate adenosine deaminase (E-ADA) activity in sera of lambs experimentally infected with Haemonchus contortus. We used 12 lambs divided into 2 groups; Group A had 5 healthy, non-infected animals (control) and Group B had 7 healthy animals infected with H. contortus . Lambs were infected orally with 500 larvae (L3) per animal every 2 days, for a period of 20 days, and later the infection was confirmed by examination of feces (eggs per gram [EPG] via fecal egg count). Blood collection was performed at days 0, 20, 40, 60, and 80 post-infection (PI) for analysis of E-ADA activity. Animals in Group A showed negative EPG throughout the experiment unlike those from Group B that had elevated EPG counts. E-ADA activity was reduced in the serum of animals infected with H. contortus when compared to non-infected controls at days 20, 40, 60, and 80 PI. Therefore, it is concluded that infection with H. contortus influences the E-ADA activity in lambs.

Comparative study of the commonly used virulence tests for laboratory diagnosis of ovine footrot caused by Dichelobacter nodosus in Australia.

Footrot in sheep and goats is expressed as a spectrum of clinical entities ranging from benign, which is a self limiting interdigital dermatitis to highly virulent, in which severe under running of the horn of the hoof occurs. Interactions between the host, the virulence of the causative strain of Dichelobacter nodosus and environmental conditions determine the severity of the disease. Clinical diagnosis of virulent footrot, which a notifiable disease in some states of Australia, is not always straightforward. Therefore, the gelatin gel and elastase tests for protease activity, and the intA PCR test for an inserted genetic element in D. nodosus are commonly used to support or to confirm a clinical diagnosis. A comparative study of these laboratory tests with a large number of samples collected from 12 flocks of sheep with clinically virulent footrot was conducted. Based on the elastase test, 64% of the isolates tested were classified as virulent compared to 91% on the gelatin gel test and 41% according to the intA test. The agreement between the elastase and the gelatin gel test was low (kappa=0.12) as were the agreements between other tests. Only about 21% of the isolates were virulent in all 3 tests. Therefore these tests on their own may not provide standard and reliable results and are likely to remain as supplementary tests for clinical diagnosis of the disease.

Responsiveness of prehaemolytic copper poisoning in sheep from a specific pathogen-free environment to a relatively high dose of tetrathiomolybdate.

Efficacy of ammonium tetrathiomolybdate (3.4 mg/kg LW, TTM(3.4)) was monitored in nine, specific pathogen-free sheep with mild-to-severe, prehaemolytic copper poisoning (pre-HCP). Five sheep were given three subcutaneous injections over seven days and four began a shorter, five-day course four days later. Plasma bile acid (BA) and glutamate dehydrogenase (GDH) had fallen significantly after six days but BA briefly rose again between days 10 to 18. Aspartate aminotransferase (AST), δ-glutamyl transferase (GGT) were later to decline but presented little evidence of hepatotoxicity by day 45. Erythrocyte superoxide dismutase (ESOD) was rapidly inhibited, activities falling by 65 per cent within three days and taking 25 days to recover. Trichloroacetic (TCA)-insoluble copper increased by 6 to 8 µmol/l after three days but had largely disappeared by day 18. Six lambs had become hypercupraemic by day 18 due to a rise in TCA-soluble Cu. ESOD activity fell again by 188 ± 29.5 U/gHb between days 25 and 45 but had largely recovered by day 62. TTM(3.4) gave better control of hepatotoxicity than TTM(1.7) had done in more severely affects cohorts but at the expense of greater inhibition of ESOD. Treatment of pre-HCuP should rely more on reducing copper absorption until more is known of the side effects of TTM on cuproenzymes.

Overexpression of aldolase A and cytokeratin 19 in ovine pulmonary adenocarcinoma.

Ovine pulmonary adenocarcinoma (OPA) is a transmissible lung cancer of sheep caused by jaagsiekte sheep retrovirus (JSRV). In the present study the protein profiles of five neoplastic and three non-neoplastic sheep lung tissues were examined for the identification of proteins overexpressed in ovine pulmonary adenocarcinoma. Lung sections of the experimental group of sheep were collected during necropsies for proteomic and immunohistochemical examination. Two dimensional electrophoresis (2DE) was performed using gel strips with immobilized pH gradient 3-10. As a result of 2DE gel analysis 14 spots characterized by over 2-fold higher expression in tumour proteomes were selected for mass spectrometry. In eleven spots more than one polypeptide was identified indicating overlapping of proteins in gels. In two spots demonstrating over 3-fold higher expression in OPA proteomes, single proteins: cytokerarin 19 (CK19) and aldolase A were identified. Immunohistochemical studies revealed that CK19 and aldolase A were expressed in the cytoplasm of epithelial cells of bronchioles in non-neoplastic lung sections, as well as epithelial cells of bronchioles and neoplastic cells in lung sections of OPA affected sheep. The results indicate that the overexpression of the two proteins reflects the presence of neoplastic cells in the lungs of OPA affected sheep.

Matrix metalloproteinase expression in sheep with listerial meningoencephalitis.

Matrix metalloproteinases (MMPs) have been implicated in the pathogenesis of several central nervous system (CNS) diseases. In this study, we investigated the presence of Listeria monocytogenes antigens and detected the expression of MMP-9 and MMP-7 in the brains of 22 sheep with clinical signs and histopathological findings characteristic of listerial meningoencephalitis. Archived sections from the brainstem, cerebrum, and cerebellum were stained for immunohistochemistry. L. monocytogenes antigens were located mainly in the cytoplasm of neutrophils and some macrophages and/or extracellularly within microabscesses of the brainstem. MMP-9 was mainly immunolocalised in the endothelial cells, microglial cells, and neurons especially in inflammatory areas. MMP-7 immunoreactivity was detected in perivascular cuffs, microglial cells, and only a few neurons. Overall, immunohistochemistry in formalin-fixed, paraffin-embedded tissues is a useful tool for the diagnosis of encephalitic listeriosis caused by L. monocytogenes, and MMP-9 and MMP-7 may contribute to the pathogenesis of listerial meningoencephalitis.

Normal vitamin D receptor function with increased expression of 25-hydroxyvitamin D(3)-24-hydroxylase in Corriedale sheep with inherited rickets.

A likely inherited disease with gross and microscopic features of rickets has been recognised in Corriedale sheep in New Zealand, and a defect in end-organ responsiveness to vitamin D has been proposed as a likely mechanism. The aim of the present study was to characterize the mode of inheritance and determine the disease mechanism. Breeding trials showed that the mode of inheritance was autosomal recessive. Serum chemistry testing using different methodology and studies in cultured skin fibroblasts did not support our previous hypothesis of a defect in end-organ responsiveness. The studies revealed normal serum 1,25-dihydroxyvitamin D concentrations, normal vitamin D receptor function, and the presence of 24-hydroxylase mRNA in cells from affected sheep, even without induction by 1,25-dihydroxyvitamin D(3). In addition, osteocalcin mRNA expression was similar in both affected and control sheep. It was concluded that increased expression of 25-hydroxyvitamin D(3)-24-hydroxylase, the enzyme that breaks down vitamin D, may be involved in the pathogenesis of inherited rickets in Corriedale sheep, but its role requires further clarification.

The subtilisin-like protease AprV2 is required for virulence and uses a novel disulphide-tethered exosite to bind substrates.

Many bacterial pathogens produce extracellular proteases that degrade the extracellular matrix of the host and therefore are involved in disease pathogenesis. Dichelobacter nodosus is the causative agent of ovine footrot, a highly contagious disease that is characterized by the separation of the hoof from the underlying tissue. D. nodosus secretes three subtilisin-like proteases whose analysis forms the basis of diagnostic tests that differentiate between virulent and benign strains and have been postulated to play a role in virulence. We have constructed protease mutants of D. nodosus; their analysis in a sheep virulence model revealed that one of these enzymes, AprV2, was required for virulence. These studies challenge the previous hypothesis that the elastase activity of AprV2 is important for disease progression, since aprV2 mutants were virulent when complemented with aprB2, which encodes a variant that has impaired elastase activity. We have determined the crystal structures of both AprV2 and AprB2 and characterized the biological activity of these enzymes. These data reveal that an unusual extended disulphide-tethered loop functions as an exosite, mediating effective enzyme-substrate interactions. The disulphide bond and Tyr92, which was located at the exposed end of the loop, were functionally important. Bioinformatic analyses suggested that other pathogenic bacteria may have proteases that utilize a similar mechanism. In conclusion, we have used an integrated multidisciplinary combination of bacterial genetics, whole animal virulence trials in the original host, biochemical studies, and comprehensive analysis of crystal structures to provide the first definitive evidence that the extracellular secreted proteases produced by D. nodosus are required for virulence and to elucidate the molecular mechanism by which these proteases bind to their natural substrates. We postulate that this exosite mechanism may be used by proteases produced by other bacterial pathogens of both humans and animals.

Tay-Sachs disease in Jacob sheep.

Autopsy studies of four Jacob sheep dying within their first 6-8 months of a progressive neurodegenerative disorder suggested the presence of a neuronal storage disease. Lysosomal enzyme studies of brain and liver from an affected animal revealed diminished activity of hexosaminidase A (Hex A) measured with an artificial substrate specific for this component of ß-hexosaminidase. Absence of Hex A activity was confirmed by cellulose acetate electrophoresis. Brain lipid analyses demonstrated the presence of increased concentrations of G(M2)-ganglioside and asialo-G(M2)-ganglioside. The hexa cDNA of Jacob sheep was cloned and sequenced revealing an identical number of nucleotides and exons as in human HexA and 86% homology in nucleotide sequence. A missense mutation was found in the hexa cDNA of the affected sheep caused by a single nucleotide change at the end of exon 11 resulting in skipping of exon 11. Transfection of normal sheep hexa cDNA into COS1 cells and human Hex A-deficient cells led to expression of Hex S but no increase in Hex A indicating absence of cross-species dimerization of sheep Hex α-subunit with human Hex ß-subunits. Using restriction site analysis, the heterozygote frequency of this mutation in Jacob sheep was determined in three geographically separate flocks to average 14%. This large naturally occurring animal model of Tay-Sachs disease is the first to offer promise as a means for trials of gene therapy applicable to human infants.

The role of lactate dehydrogenase, alkaline phosphatase and aspartate aminotransferase in the diagnosis of subclinical intramammary infections in dairy sheep and goats.

The objective was to investigate the changes occurring in the activities of the enzymes lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and aspartate aminotransferase (AST) in sheep and goat milk as a result of subclinical intramammary infections (IMI) and to evaluate the use of these enzymes for the diagnosis of subclinical IMI in dairy sheep and goats. A total of 206 samples of sheep milk and 162 samples of goat milk, obtained from equal udder halves, were used in the study. For each species they were divided into two groups: a no-infection group and a subclinical infection group. Activities of LDH, ALP and AST were significantly higher in the subclinical infection group than in the no-infection group (P<0.05) in both sheep (LDH: 350.42+/-11.25 v. 120.91+/-4.41; ALP: 2773.43+/-105.18 v. 2189+/-94.24; AST: 29.57+/-0.74 v. 17.32+/-0.46) and goats (LDH: 354.07+/-13.33 v. 103.79+/-3.75; ALP: 311.13+/-25.74 v. 137.24+/-19.62; AST: 27.59+/-6.42 v. 15.87+/-0.45). The activity of LDH was identified as indicator for subclinical IMI in both sheep and goats. The optimum cut-off values for LDH activity, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic (ROC) analysis, were at 197 U/l, 185 U/l and 197 U/l for sheep, goats and both species, respectively. DSn for sheep, goats and both species at these cut-off values was 92.8%, 98.2% and 94.0%, whereas DSp was 95.4%, 96.3% and 96.3%, respectively. It was concluded that the determination of LDH activity in milk serum is a sensitive and reliable method for the detection of subclinical IMI in dairy sheep and goats.

Metabolic acidosis in sheep alters expression of renal and skeletal muscle amino acid enzymes and transporters.

To determine the effect of metabolic acidosis on expression of L-Gln, L-Glu, and L-Asp metabolizing enzymes and transporters, the relative content of mRNA, protein, or mRNA and protein, of 6 enzymes and 5 transporters was determined by real-time reverse transcription-PCR and immunoblot analyses in homogenates of kidney, skeletal muscle, and liver of growing lambs fed a common diet supplemented with canola meal (control; n = 5) or HCl-treated canola meal (acidosis; n = 5). Acidotic sheep had a 790% greater (P = 0.050) expression of renal Na(+)-coupled neutral AA transporter 3 mRNA and a decreased expression of renal glutamine synthetase mRNA (47% reduction, P = 0.037) and protein (57% reduction, P = 0.015) than control sheep. No change in renal cytosolic phosphoenolpyruvate carboxykinase (protein and mRNA), glutaminase (mRNA), or L-Glu dehydrogenase (protein) was found. In skeletal muscle, acidotic sheep had 101% more (P = 0.026) aspartate transaminase protein than did control sheep, whereas no change in the content of 3 Na(+)-coupled neutral AA transporters (mRNA) or 2 high-affinity L-Glu transporter proteins was found. In liver, no change in the content of any assessed enzyme or transporter was found. Collectively, these findings suggest that tissue-level responses of sheep to metabolic acidosis are different than for nonruminants. More specifically, these results indicate the potential capacity for metabolism of L-Asp and L-Glu by skeletal muscle, and L-Gln absorption by kidneys, but no change in hepatic expression of L-Gln metabolism, elaborates previous metabolic studies by revealing molecular-level responses to metabolic acidosis in sheep. The reader is cautioned that the metabolic acidosis model employed in this study differs from the increased plasma lactate-induced metabolic acidosis commonly observed in ruminants fed a highly fermentable grain diet.

Deletion of the C-terminus of polynucleotide phosphorylase increases twitching motility, a virulence characteristic of the anaerobic bacterial pathogen Dichelobacter nodosus.

The Gram-negative anaerobe Dichelobacter nodosus is the causative agent of footrot in sheep. Different strains of D. nodosus cause disease of differing severities, ranging from benign to virulent. Virulent strains have greater twitching motility and secrete proteases that are more thermostable than those secreted by benign strains. We have identified polynucleotide phosphorylase (PNPase) as a putative virulence regulator and have proposed that PNPase expression is modulated by the adjacent integration of genetic elements. In this study, we compared PNPase activity in three virulent and four benign strains of D. nodosus and found that PNPase activity is lower in virulent strains. We disrupted the pnpA gene in three benign D. nodosus strains and two virulent strains and showed that deletion of the S1 domain of PNPase reduced catalytic activity. In all but one case, deletion of the PNPase S1 domain had no effect on the thermostability of extracellular proteases. However, this deletion resulted in an increase in twitching motility in benign, but not in virulent strains. Reconstruction of the pnpA gene in two mutant benign strains reduced twitching motility to the parental level. These results support the hypothesis that PNPase is a virulence repressor in benign strains of D. nodosus.

Serum T3 and T4 concentrations in lambs with nutritional myodegeneration.

BACKGROUND: Selenium (Se) deficiency is an important etiological factor in Nutritional Myodegeneration Disease (NMD) of lambs, and Se is required for synthesis of thyroid hormones. HYPOTHESIS: That serum T4 and T3 concentrations in lambs with NMD will be abnormal. ANIMALS: Ten healthy lambs and 15 lambs with NMD were included in the study. METHODS: Serum thyroid hormone concentrations were measured by chemiluminescence assay in samples collected from control and NMD lambs before and after treatment with a preparation containing sodium selenite, vitamin E, and vitamin B1, which was administered subcutaneously to lambs with NMD twice, with a 2-week interval. RESULTS: Before treatment, serum concentrations of fT3 and TT3 in lambs with NMD were lower than those of control group (P < .001), while serum concentrations of fT4 and TT4 in lambs with NMD were significantly higher than those of control group (P < .001, P < .01, respectively). Similarly, serum creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) values in lambs with NMD were significantly higher compared with those of control (P < .001, P < .001, P < .001, P < .01, and P < .05, respectively) before treatment. However, serum concentrations of Se and alpha-tocopherol in lambs with NMD were significantly lower compared with those of control (P < .001). After treatment (at day 30), none of the variables were significantly different between control and NMD lambs (P > .05). CONCLUSIONS AND CLINICAL IMPORTANCE: Nutritional myodegeneration in lambs is associated with abnormalities in serum thyroid concentrations. Abnormalities in serum thyroid concentrations can result from selenium deficiency in NMD lambs. Serum thyroid concentrations together with serum CK, LDH, AST, ALT, and ALP values can be considered additional tools in diagnosis and prognosis of NMD in lambs.

Selected trace elements and esterase activity of carbonic anhydrase levels in lambs with pneumonia.

The levels of, zinc, copper, Fe, Zn, Cu, Mn, Mg, K, Na, and Cl and the activity of carbonic anhydrase were determined in lambs with pneumonia. A significant decrease of p < 0.01 level in Zn concentration, in Cu level (p < 0.001) and significant increases in K and Na levels (p < 0.05) and of the Cu/Zn ratio (p < 0.001) were observed in the study group. The carbonic anhydrase activity was decreased in the study group, but the decrease was not statistically significant (p > 0.05). Also, nonsignificant decreases of Fe, Mg, and Cl and increase of the Mn concentration were also observed in the lambs with pneumonia (p > 0.05). Our results suggest that the significant element changes reported here and the Cu/Zn ratio, but not the activity of carbonic anhydrase, can be used as indicators of pneumococcal infection.

Defective gamma-glutamyl carboxylase activity and bleeding in Rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality caused by ineffective hemostasis at parturition. Neonatal-affected lambs presented with inadequate hemostasis at the umbilicus, pale mucus membranes, and markedly prolonged activated clotting time. Affected lambs had consistently prolonged 1-stage prothrombin times and activated partial thromboplastin times that supported a defect in the common pathway or defects in both the intrinsic and extrinsic pathway of the coagulation cascade. Decreased activity of vitamin K-dependent procoagulant factors II, VII, IX, and X in male and female lambs suggested either a defect of the hepatic enzyme gamma-glutamyl carboxylase, or vitamin K(1) 2,3 epoxide reductase. Affected lamb hepatic gamma-glutamyl carboxylase activity was markedly decreased compared with that of age- and sex-matched control lambs, while vitamin K(1) 2,3 epoxide reductase and glucose-6-phosphatase activities were similar between an affected and normal lamb. Subcutaneous vitamin K(1) supplementation did not increase vitamin K-dependent procoagulant factor activities in 3 lambs administered vitamin K(1) daily. These data confirm defective gamma-glutamyl carboxylase activity as the cause of impaired coagulation of sheep in this flock. This flock represents the only viable animal model of hereditarily defective gamma-glutamyl carboxylase activity.

Truncated gamma-glutamyl carboxylase in rambouillet sheep.

A flock of Rambouillet sheep was examined because of increased lamb mortality due to ineffective hemostasis at parturition. Decreased activities of coagulation factors II, VII, IX, and X, and severely reduced hepatic gamma-glutamyl carboxylase activity with adequate vitamin K 2,3 epoxide reductase activity was determined.(1,)(21) Parenteral vitamin K(1) supplementation did not improve vitamin K-dependent coagulation factor activities in 3 affected lambs. Affected lamb gamma-glutamyl carboxylase deoxyribonucleic acid was sequenced, and 4 single nucleotide polymorphisms (SNPs 2-5) of the gamma-glutamyl carboxylase gene were identified. Single nucleotide polymorphism-4 results in an arginine to stop codon (UGA) substitution, which prematurely terminates the peptide at residue 686 (R686Stop). This genotype (GATT/GATT) has a strong association with the coagulopathy observed in clinically affected lambs, P < 0.001. The frequency of SNP-3 in exon 11 (R486H) within the MARC 1.1 database is high in the US sheep population overall. Gamma-glutamyl carboxylase activity in hepatic microsomes from a SNP-3 homozygous lamb lacking the SNP-4 mutation (GACC/GACC) was similar to control sheep homozygous for arginine at 486 and also lacking SNP-4 (TGCC/TGCC), indicating that the R486H does not measurably impact gamma-glutamyl carboxylase activity. The remaining two SNPs (2 and 5) are located within non-coding intron sequences. These 4 SNPs allowed for determining the genotype associated with the observed fatal coagulopathy. Screening for the premature truncation (SNP-4) based on the presence of a Bbv I restriction site in clinically normal lambs but not in the homozygous affected lambs allows for detection of the heterozygous state (GATT/GACC), because carrier animals are clinically normal.

Clearance of para-aminohippuric acid in wethers consuming locoweed.

AIM: To validate the use of para-aminohippuric acid (PAH) as a marker for measuring blood flow in wethers consuming a mixed diet of locoweed and blue grama hay. METHODS: Fourteen sheep, stratified by bodyweight (BW), were assigned to one of three treatments: 0.8 mg swainsonine (SW)/kg BW (HI), 0.2 mg SW/kg BW (LO), and no SW (Control). Sheep were fed various ratios of locoweed and blue grama hay to deliver SW treatments, for 28 days prior to infusion of PAH. Concentrations of SW and activities of alkaline phosphatase (Alk-P) and aspartate aminotransferase (AST) in serum were measured to confirm exposure to SW and subclinical intoxication. A single 20-ml injection of 5% PAH was delivered into the jugular vein after subclinical intoxication had been achieved. Blood samples were collected and serum analysed for PAH immediately prior to injection, then every 5 min from 5-30 min, and every 10 min from 30-60 min, following injection of PAH. RESULTS: Effective delivery of SW was evident from the greater concentrations of SW measured in the serum of HI compared with LO animals (p<0.05). No significant differences were detected in the rate of elimination (range 0.097-0.108 L/min), elimination half-life (range 6.62-7.24 min), apparent volume of distribution for the central compartment (range 7.14-9.72 L), and clearance (range 0.73-0.92 L/min) of PAH, between treatments. CONCLUSIONS: Subclinical intoxication with SW did not affect the pharmacokinetics of PAH. Thus, use of downstream dilution of PAH is a valid method to determine the rate of blood flow in nutrient flux experiments that involve consumption of locoweed.