Ovine Urine Research Articles

Evaluation of Heller's reaction and the urine dipstick method alone or in combination to detect proteinuria in sheep

Urine dipstick and Heller's reaction are easy first-line screening tests for the detection of proteinuria; however, the performance of these methods in alkaline ovine urine is largely unknown. The aim of this study was to evaluate the diagnostic performance of Heller's reaction alone or in combination with dipstick for the detection of proteinuria in sheep, using the urine protein to creatinine ratio (UP/C) with two cut-off values as the reference method. Ninety-eight urine samples were collected from sheep using the transient apnea method. Heller's reaction, the dipstick method, and the UP/C ratio were used to assess proteinuria. The results were statistically analyzed twice, based on two different UP/C cut-off values of 0.2 and 0.5. Cohen's kappa value was used to determine the agreement between the UP/C ratios and Heller's reaction, the dipstick method, or the combination of methods. Sensitivity, specificity, and positive and negative likelihood ratios were calculated. ROC curves were also generated, and the areas under the curve (AUC) were evaluated to determine the optimal threshold for the numerical values of the two methods. Heller's reaction is more specific (96.67% and 96.00% when the cut-off value is 0.2 and 0.5, respectively) than the dipstick method, while the dipstick method was more sensitive (91.18% and 91.30%, when the cut-off value was 0.2 and 0.5, respectively) than Heller's reaction for the detection of proteinuria. Both tests were accurate when any grade >0 was considered positive. Proteinuria can almost be excluded in ovine urine samples with negative Heller's reaction and dipstick test.

Effect of storage time on the urine protein: creatinine ratio in alkaline ovine urine.

The urine protein:creatinine (UPC) ratio is considered the reference method to assess proteinuria. Its diagnostic value in ovine medicine needs further elucidation. In population monitoring and/or for research purposes, it is convenient to collect many samples simultaneously and store them for later analysis. However, analyte stability data are required to ensure reliable results. We used 15 of 90 urine samples collected from sheep to assess the effect of storage time on the UPC ratio. After centrifugation, the supernatant of each sample was divided into 6 aliquots. Urine protein and creatinine concentrations were determined immediately in one aliquot using the pyrogallol red and a modified Jaffè method, respectively. The other aliquots were stored at -18°C. Based on the absence of active sediment, alkaline urine pH, and UPC ratio ≥0.2, we included 15 samples in our study. The UPC ratio was determined in the stored aliquots 2, 7, 14, 21, and 60 d after collection. The data were analyzed with univariate ANOVA. No significant difference was observed in the urinary concentrations of protein, creatinine, and the UPC ratio (0.8 ± 0.84 in conventional units and 0.09 ± 0.095 in SI units) among different times (p > 0.05). The UPC ratio remained stable for 2 mo in ovine urine samples stored at -18°C.

Real-time PCR for detection of Brucella ovis and Histophilus somni in ovine urine and semen

A epididimite infecciosa ovina e uma das principais enfermidades reprodutivas de carneiros. O presente estudo teve por objetivo desenvolver protocolos de PCR em tempo real para B. ovis e H. somni e avaliar sua aplicabilidade em amostras de semen e urina de carneiros. Delinearam-se primers e sondas especie-especificos para cada agente. As sondas foram delineadas com o sistema TaqMan incorporando um marcador FAM para B. ovis e Cy5 para H. somni na extremidade 5' e um quencher na extremidade 3'. A PCR em tempo real para B. ovis e H. somni foi altamente sensivel, uma vez que a amplificacao de DNA ocorreu com ate 0,2ng de DNA/reacao. A especificidade dos iniciadores e sondas foi avaliada com amostras de DNA de outros agentes causadores de epididimite ovina e nenhuma amplificacao inespecifica foi observada. A aplicabilidade da tecnica em amostras biologicas tambem foi confirmada, pois nao houve perda de eficacia (P>0,05) quando comparada a PCR convencional com amostras de semen e urina de carneiros experimentalmente infectados.

Metabolic pathways of anthelmintic drug monepantel in sheep and in its parasite (Haemonchus contortus)

Monepantel (MOP) is a new anthelmintic drug intended for the treatment and control of gastrointestinal roundworms (nematodes) infection and associated disease in sheep. The aim of our study was to find out metabolic pathways of MOP in sheep in vivo and in its parasite Haemonchus contortus ex vivo. MOP biotransformation in two H. contortus strains with different sensitivity to anthelmintics was also compared. Ultra high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) technique is used for the identification of MOP metabolites in ovine urine, faeces, and nematodes. MOP biotransformation study in sheep in vivo led to the identification of 13 MOP metabolites; 7 of them have not been described previously in in vitro study. The study of MOP biotransformation in H. contorus ex vivo reveals four MOP metabolites. The nitrile hydrolysis as a new biotransformation pathway in helminths ex vivo is reported here for the first time. Unlike sheep, H. contorus nematodes are not able to metabolize MOP via phase II biotransformation. Nematodes of resistant White river (WR) strain form more types of MOP metabolites than nematodes of sensitive inbred susceptible Edinburgh (ISE) strain. Based on obtained results, schemes of metabolic pathways of MOP in sheep and nematodes are proposed.

Comparison of Shedding, and Antibiotic Resistance Properties of Listeria monocytogenes Isolated From Milk, Feces, Urine, and Vaginal Secretion of Bovine, Ovine, Caprine, Buffalo, and Camel Species in Iran

Background: Listeria monocytogenes causes listeriosis characterized by encephalitis, septicaemia, and abortion or stillbirth. Its traditional diagnosis is based on serological responses, whileseveral molecular methods have been developed for safer and more rapid, sensitive, and accurate detection.The epidemiology, prevalence, shedding routes, and antibiotic resistance properties of L. monocytogenes are essentially unknown in various animals species. Objectives: The present study was performed to study the shedding routes, and antibiotic resistance properties of L. monocytogenes isolated from milk, feces, urine, and vaginal secretion of bovine, ovine, caprine, buffalo, and camel in Iran. Materials and Methods: A total of 596 milk, 619 feces, 443 vaginal swab, and 522 urine samples were collected from various animal species. Samples were examined by culture, conventional and real-time PCR for evaluating the presence of L. monocytogenes. Finally antimicrobial resistance properties were studied using the simple disc diffusion method. Results: The culture method showed that 186 of 2180 samples (8.53%) had positive results for L monocytogenes. In total,61 (10.23%) milk, 40 (6.46%) feces, 43 (9.7%) vaginal swab, and 48 (9.19%) urine samples had positive results for L monocytogenes using conventional PCR. After the Light Cycler real-time PCR it was recognized that 69 (11.57%) milk, 48 (7.75%) feces, 53 (11.96%) vaginal swab and 57 (10.91%) urine samples hadpositive results for the presence of L. monocytogenes. The sensitivity and specificity of conventional and real-time PCR were 94% and 99.1%, and 100% and 97.9%, respectively. L. monocytogenes had the highest shedding in bovine milk (10.83%), ovine urine (16.98%), caprine feces (14.38%), buffalo milk (11.11%), and camel vaginal secretion (15.18%). Antibiotic resistance to tetracycline (71.3%) was the highest, while the resistance to nitrofurantoin (5.72%) had the lowest frequency. Conclusions: Shedding of L. monocytogenes in different animal species, and different samples are different. Due to antibiotic resistance, especially in L. monocytogenes, veterinarians should pay more attention to prescribe antibiotics. We recommend using the real-time PCR for safe, sensitive, and rapid detection of L. monocytogenes in clinical samples, and using the disk diffusion methods to prescribe suitable antibiotics.

Description of a novel viral tool to identify and quantify ovine faecal pollution in the environment

Farmed animals such as sheep, cattle, swine and poultry play an important role in microbial contamination of water, crops and food, and introduce large quantities of pathogens into the environment. The ability to determine the origin of faecal pollution in water resources is essential when establishing a robust and efficient water management system. Animal-specific viruses have previously been suggested as microbial source tracking tools, but specific ovine viral markers have not been reported before now. Previous studies have shown that polyomaviruses are host-specific, highly prevalent and are commonly excreted in urine. Furthermore, they have been reported to infect several vertebrate species but not sheep. That situation encouraged the study of a new putative ovine polyomavirus (OPyV) and its use to determine whether faecal pollution originates from ovine faecal/urine contamination.Putative OPyV DNA was amplified from ovine urine and faecal samples using a broad-spectrum nested PCR (nPCR). Specific nested PCR and quantitative PCR assays were developed and applied to faecal and environmental samples, including sheep slurries, slaughterhouse wastewater effluents, urban sewage and river water samples. Successful amplification by PCR was achieved in sheep urine samples, sheep slaughterhouse wastewater and downstream sewage effluents. The assay was specific and was negative in samples of human, bovine, goat, swine and chicken origin. Ovine faecal pollution was detected in river water samples by applying the designed methods. These results provide a quantitative tool for the analysis of OPyV as a suitable viral indicator of sheep faecal contamination that may be present in the environment.

Laboratory diagnostics in the urine of young and pregnant ewes

The purpose of this study was to identify values for net acid base excretion (NABE) which are significant indicators of the acid-base equilibrium in pregnant and young ewes and to show its relationship with other parameters (base, acid, ammonium [NH4], base-acid quotient, sodium [Na], potassium [K], calcium [Ca]) in ovine urine. In contrast to dairy cows, data are rare on these parameters in ewes. A total of 99 animals were used in the study, consisting of 56 young (average of 5.6±1.1 months) and 43 pregnant ewes (average of 35.2±18.8 months). Measurement of fractional NABE in urine samples was carried out according to the method reported by Kutas. The pH value of the urine was measured with a laboratory pH meter. Na, K and total Ca were measured with a flame photometer. For all values except Na significant differences occurred between urine samples of pregnant ewes and young ewes (p<0.001). Base, acid, NH4, NABE, K and Ca values were significantly higher in the urine of the youngs than in pregnant ewes. In young ewes, a strong correlation was found between NABE and base values while a weak correlation could be observed between pH and base values. In pregnant ewes, strong NABE-base, NABE-K, K-acid and K-base correlations were found as well as weak NH4-base, NH4-NABE and NH4-K correlations. There was a strongly positive correlation between NABE and NH4 in pregnant ewes, while a weak negative correlation between those values was observed in young ewes. For the first time, we established values for NABE and certain other parameters in urine of pregnant ewes and young ewes. It was shown that the acid-base balance in pregnant ewes and young ewes can be evaluated by measuring NABE and certain trace elements in urine like in cattle.

A highly efficient method for determination of allantoin in ruminal digesta, ovine urine and blood serum by HPLC

A sensitive HPLC method for direct determination of allantoin in ruminal digesta, urine and blood serum of sheep is described. Blood serum and urine samples were diluted 1:3 with deionized water. Separation and quantification of allantoin was achieved using three long Nova Pac C 18 -co- lumns (Waters) and UV detection at 205 and 215 nm. The low coefficient of variation and satisfac- tory recovery of allantoin added to assayed samples (~100%) provide a suitable tool for routine quantification of allantoin in a large number of urine, ruminal digesta and blood serum samples.

Simultaneous determination of purine metabolites in ovine urine and blood plasma by high-performance liquid chromatography

1. Effects of fertiliser nitrogen rate to spring grass on apparent digestibility, nitrogen balance, ruminal fermentation and microbial nitrogen production in beef cattle and in vitro rumen fermentation and methane output A. O’Connor, A.P. Moloney, P. O’Kiely, T. Boland, M. McGee Animal Feed Science and Technology CrossRef

Determination of grayanotoxins in biological samples by LC-MS/MS.

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.

Quantification of allantoin, uric acid, xanthine and hypoxanthine in ovine urine by high-performance liquid chromatography and photodiode array detection

A HPLC method for the determination of allantoin, uric acid, hypoxanthine and xanthine (purine metabolites) in ovine urine without the disadvantages inherent in derivatization is described. After dilution 1:6 with water, urine samples were injected onto the column. Separation and quantification of purine metabolites was achieved using two Nova-Pak C 18 columns (4 μm, 250×4.6 mm, Waters). A binary gradient program and UV detection were used for purine metabolites analysis. Clear resolution of purine metabolites was obtained in less than 15 min. Allantoin, uric acid, hypoxanthine and xanthine in the effluent were monitored at 225, 284, 250 and 267 nm, respectively. The average recoveries of purine metabolite standards added to urine samples were satisfactory (100.2–102.9%). The low coefficients of variation (0.29–0.73%) as well as the low detection (0.16–0.70 nmol) and quantification (0.52–2.32 nmol) limits indicate satisfactory precision, reproducibility and sensitivity of the proposed method. This procedure is suitable for routine quantification of purine metabolites in a large number of urine samples.

Simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine by reversed-phase high-performance liquid chromatography

Determination of purine metabolites, pseudouridine and creatinine in both bovine and ovine urine using high-performance liquid chromatography (HPLC) is described. Following dilution and filtration, urine samples were analysed directly. Separation and quantification was achieved using a Spherisorb ODS II C 18 column (250×4.6 mm I.D.) under isocratic conditions. The mobile phase contained 7.5 m M ammonium dihydrogen phosphate, 10 m M sodium 1-heptane sulphonic acid and 1.0 m M triethylamine at pH 3.0. Chromatography was achieved at a flow-rate of 1.0 ml/min and monitoring column effluent at 218 nm. Total analysis time was 60 min. Recovery of all compound standards added to urine was above 96%. In all cases, close spectral matches of compound standards and corresponding identified peaks in ovine and bovine urine were obtained. Lowest detectable concentrations of allantoin, uric acid, xanthine, hypoxanthine, creatinine and pseudouridine were 1.1, 1.0, 1.0, 1.0, 3.0 and 0.4 μmol/l, respectively. Advantages of simultaneous determination of purine metabolites, creatinine and pseudouridine in ruminant urine collected from both sheep and cattle exist over current methods.

Bank of reference samples of blank urine from livestock

From three species of livestock, bovine, ovine and porcine, samples of urine from each 10–20 different animals were collected. The animals originated from a governmental experimental farm and differed in race, age, sex and were fed with different types of feed. Fifty different samples of urine were lyophilised in units of 5 mL in sealed amber glass bottles. After lyophilisation of the samples, several quality control tests were performed. The variation in net weight of the units of urine and the lyophilisates ranged from 0.01%–1.7% and 0.1%–10%, respectively. Most values did not exceed 0.1% for urines and 1% for lyophilisates. The residual water content of the lyophilisates was determined by the Karl-Fisher titration method. The average percentage of residual water ranged from 1.0%–7.0% for bovine, from 1.3%–3.0% for porcine and from 1.6%–5.5% for ovine urine lyophilisates. Further, all different samples were analysed for the presence of anabolic compounds with a multi-residue procedure developed at the CRL. In two samples of bovine urine the presence of α-boldenone was detected and confirmed. In most porcine urines the endogenous steroids α-testosterone and α-estradiol were detected. In a number of porcine and ovine urine samples α- and β-zearalenol were detected. The origin of these compounds is the f2-toxin produced by a Fusarium fungus. In ovine samples α-testosterone and α-estradiol were detected.

Possible naturally occurring zeranol in bovine bile in Northern Ireland.

Zeranol and two Fusarium toxins, alpha-zearalenol and beta-zearalenol, were confirmed by gaschromatography/mass spectrometry (GC/MS) in bovine bile samples referred to this laboratory for analysis. No evidence of zeranol abuse was found on-farm. Given the recent suggestion that zeranol might arise from the metabolism of these Fusarium toxins, and the finding of zeranol in bovine and ovine urine across the EU, it was concluded that the residues had arisen as a result of natural metabolism.

Development of an Enzyme-Linked Immunosorbent Assay for Analysis of Sporidesmin A and Its Metabolites in Ovine Urine and Bile

Monoclonal and polyclonal antibodies have been produced for the development of competitive enzymelinked immunosorbent assays for sporidesmin A, the mycotoxin that causes facial eczema in grazing livestock. Hemisuccinyl derivatives of sporidesmin conjugated to a number of proteins were used as immunizing and coating antigens. Two immunoassays have been developed. Each detects the presence of a different region of the sporidesmin molecule, therefore allowing the two major sporidesmins found in grass or in culture to be distinguished as well as allowing the detection of metabolites in ovine urine and bile. An analytical procedure that did not include organic solvent extraction was developed so that water-soluble metabolites would also be detected

Effect of ovine urine and some of its components on viability of nematode eggs and larvae in sheep faeces

Sheep faeces incubated for 7 days at 27°C for cultivation of third-stage nematode larvae were sprinkled daily with urine from sheep or with solutions of components normally occurring in sheep urine. Larval development was completely blocked in cultures sprinkled either with sheep urine, with solutions of 2 or 4% urea, or with urine from which urea or the phenol components had been extracted. Only a few third-stage larvae developed in cultures sprinkled with 1% urea. Normal larval development occurred in cultures sprinkled with either the phenol component from urine, or with solutions of 0.035% phenol, 0.035% p-cresol, 0.3% allantoin, 0.3% hippuric acid or 2.8% NaCl. Normal larval development also occurred in all control cultures sprinkled with water, including one culture where there was urine in the space between the outer and inner beaker used for cultivation. It is suggested that the inhibitory effect of urine on larval development is mainly caused by ammonia produced when urinary urea is brought into contact with urease of faecal origin. It is, however, an unsolved question why urine, from which urea had been removed, also inhibited larval development.

THE URINARY EXCRETION OF GONADOTROPHINS BY SHEEP: EXTRACTION PROCEDURE AND DAY-TO-DAY FLUCTUATION IN EXCRETION BY MALE AND FEMALE CASTRATED SHEEP.

SUMMARY The application of Johnsen's tannic acid method to the extraction of gonadotrophins from ovine urine was studied using the urine of castrated sheep. Slight modification of the original method increased the efficiency of extraction. Rate of recovery was measured at several levels of added pituitary and urinary gonadotrophins and was similar to recovery rates reported for human urines (30–81 %). Reproducibility of the extraction procedure was excellent. Further purification of the final ethanol precipitate without loss in potency was effected by chromatography using diethylaminoethyl cellulose. The daily excretion of total gonadotrophic activity, follicle stimulating hormone and luteinizing hormone by two male and one female castrated sheep was estimated for four periods each of 3 weeks. In all instances excretory levels fluctuated considerably and randomly. Levels of excretion of all types of gonadotrophic activity were much lower than in man.

Isolation and characterization of a urinary mucoprotein from ovine urine

Physical examination of a threadlike ovine urinary mucoprotein revealed a close similarity to Tamm and Horsfall's mucoprotein of human urine. The long and slender filaments observed by electron microscopy have widths of 150 A., lengths between 2500 and 40,000 A., and repeated beading at 200 A. The ovine mucoprotein behaved as a single component in the ultracentrifuge with a sedimentation coefficient of 30 S as found by extrapolation to zero mucoprotein concentration. At pH 7.5–7.8 the electrophoretic mobility of the mucoprotein was approximately −11.6 × 10 −5 cm. 2 sec. −1 v. −1.