Serum protein concentrations from clinically healthy horses determined by agarose gel electrophoresis

Background: Serum protein electrophoresis is a useful screening test in equine laboratory medicine. The method can provide valuable information about changes in the concentrations of albumin and α‐, β‐, and γ‐globulins and thereby help characterize dysproteinemias in equine patients. Reference values for horses using agarose gel as a support medium have not been reported. Objectives: The purpose of this study was to establish reference intervals for serum protein concentrations in adult horses using agarose gel electrophoresis and to assess differences between warm‐blooded and heavy draught horses. In addition, the precision of electrophoresis for determining fraction percentages and the detection limit were determined. Methods: Blood samples were obtained from 126 clinically healthy horses, including 105 Thoroughbreds and 21 heavy draught horses of both sexes and ranging from 2 to 20 years of age. The total protein concentration was determined by an automated biuret method. Serum protein electrophoresis was performed using a semi‐automated agarose gel electrophoresis system. Coefficients of variation (CVs) were calculated for within‐run and within‐assay precision. Data from warm‐blooded and draught horses were compared using the Mann–Whitney U test. Results: Within‐run and within‐assay CVs were <5% for all protein fractions. No significant difference was found between warm‐blooded and heavy draught horses and so combined reference intervals (2.5–97.5%) were calculated for total protein (51.0–72.0 g/L), albumin (29.6–38.5 g/L), α₁‐globulin (1.9–3.1 g/L), α₂‐globulin (5.3–8.7 g/L), β₁‐globulin (2.8–7.3g/L), β₂‐globulin (2.2–6.0 g/L), and γ‐globulin (5.8–12.7 g/L) concentrations, and albumin/globulin ratio (0.93–1.65). Conclusion: Using agarose gel as the supporting matrix for serum protein electrophoresis in horses resulted in excellent resolution and accurate results that facilitated standardization into 6 protein fractions.     

Fluorescence spectra and measurement of phylloerythrin (phytoporphyrin) in plasma from clinically healthy sheep, goats, cattle and horses

AIM: To measure the background concentration of phylloerythrin in plasma from clinically healthy sheep, goats, cattle and horses on pasture. METHODS: Blood samples were taken from 34 sheep of the Dala breed, 20 female Norwegian dairy goats, 35 Norwegian Red cows and 34 horses of different breeds. All animals were grazing green pasture when blood samples were taken. Blood samples were collected from each of four clinically healthy newborn lambs, goats, calves and foals, and pooled into one sample per species. Plasma samples were analysed for phylloerythrin by fluorescence spectroscopy, using a Perkin-Elmer LS-50B luminescence spectrometer equipped with a red-sensitive photomultiplier. The fluorescence spectra of phylloerythrin in plasma from the adult ruminants were compared with those in plasma from the neonatal ruminants, to which a known concentration of phylloerythrin had been added. RESULTS: Plasma obtained from the adult ruminants had spectral characteristics similar to those of phylloerythrin, namely weak emission peaks at 650 and 711 nm, when excited at 425 nm. Emission spectra obtained from plasma from the neonatal ruminants showed no fluorescence at these wavelengths. On average, 0.012 (SD 0.004), 0.06 (SD 0.04), and 0.05 (SD 0.03) micromol/l phylloerythrin were present in plasma samples from the sheep, goats, and cattle, respectively. The fluorescence spectra of plasma from the newborn foals were similar to spectra of plasma from adult horses, with weak emission at 669 nm. CONCLUSION: Small concentrations of phylloerythrin were detected in plasma from clinically healthy sheep, goats and cattle, but none could be detected in plasma from clinically healthy horses.     

Fluorescence spectra and measurement of phylloerythrin (phytoporphyrin) in plasma from clinically healthy sheep,goats, cattle and horses

AIM: To measure the background concentration of phylloerythrin in plasma from clinically healthy sheep, goats, cattle and horses on pasture.

METHODS: Blood samples were taken from 34 sheep of the Dala breed, 20 female Norwegian dairy goats, 35 Norwegian Red cows and 34 horses of different breeds. All animals were grazing green pasture when blood samples were taken. Blood samples were collected from each of four clinically healthy newborn lambs, goats, calves and foals, and pooled into one sample per species. Plasma samples were analysed for phylloerythrin by fluorescence spectroscopy, using a Perkin-Elmer LS-50B luminescence spectrometer equipped with a red-sensitive photo- multiplier. The fluorescence spectra of phylloerythrin in plasma from the adult ruminants were compared with those in plasma from the neonatal ruminants, to which a known concentration of phylloerythrin had been added.

RESULTS: Plasma obtained from the adult ruminants had spectral characteristics similar to those of phylloerythrin, namely weak emission peaks at 650 and 711 nm, when excited at 425 nm. Emission spectra obtained from plasma from the neonatal ruminants showed no fluorescence at these wavelengths. On average, 0.012 (SD 0.004), 0.06 (SD 0.04), and 0.05 (SD 0.03) μmol/l phylloerythrin were present in plasma samples from the sheep, goats, and cattle, respectively. The fluorescence spectra of plasma from the newborn foals were similar to spectra of plasma from adult horses, with weak emission at 669 nm.

CONCLUSION: Small concentrations of phylloerythrin were detected in plasma from clinically healthy sheep, goats and cattle, but none could be detected in plasma from clinically healthy horses.     

Characterization of Staphylococcus intermedius from pigeons, dogs, foxes, mink, and horses by pulsed-field gel electrophoresis

Staphylococcus intermedius from pigeons, dogs, foxes, mink, and horses, was characterized by pulsed-field gel electrophoresis (PFGE) to evaluate the use of this typing method for discriminating among strains. SmaI cut the chromosomal DNA into 7-13 fragments ranging from approximately 48 kb to 655 kb, with most of the detectable fragments being smaller than 172 kb. S. intermedius from various animals had a high degree of restriction fragment length polymorphism. Pigeon strains have a similar genotype, despite the difference in their isolation area. Phage typing indicated that the dog, fox, and mink strains belong to the canine I or canine II type. The PFGE method further differentiated the mink strains from the dog and fox strains with regard to three fragments between 256 kb and 570 kb. As such, genomic DNA fingerprinting by PFGE appears to be an effective technique for discriminating S. intermedius strains from various animals. A combination of PFGE typing and phage typing would provide more detailed information than the single method for ecological investigations of S. intermedius.     

Analysis of mouse, rat, dog, marmoset, and human serum proteins by capillary electrophoresis: comparison with agarose gel electrophoresis

BACKGROUND: Serum protein analysis in both humans and experimental animal species has so far been carried out by labor-intensive techniques, such as agarose gel electrophoresis (AGE). OBJECTIVE: The objective of this study was to evaluate capillary electrophoresis (CE) as an alternative technique to AGE for the analysis of serum proteins from healthy animals. METHODS: Blood samples were collected into tubes without anticoagulant from 6 fasted healthy male mice, rats, dogs, marmosets, and humans. Serum proteins were separated by CE using a technique standardized for the analysis of human proteins, and the results (efficiency, resolution, and precision) were compared with those obtained through AGE. RESULTS: Compared with AGE, CE resulted in narrower peaks and more peaks. The efficiency of protein separation by CE was significantly higher for all species, and resolution (R) was significantly higher in samples from dogs. Using rat serum, intraday reproducibility was lower for all protein fractions, and interday reproducibility was lower for most peaks, compared with AGE. CONCLUSIONS: We conclude that CE is a viable alternative to AGE for the determination of protein electrophoresis in a routine veterinary clinical pathology laboratory. The minimal sample requirement (2 microL), complete automation, and quantitative results make CE an especially valuable technique for protein analysis in experimental animal models.     

Sonographic diagnosis of early pregnancy in horses, cattle, sheep, goats, swine, dogs and cats. Standard values and limitations

Ultrasonography allows early and reliable pregnancy detection in several domestic animals. Transrectal sonography can be recommended in horses and cattle, transrectal or transcutaneous procedures in sheep, goats and pigs while transcutaneous ultrasound scanning is appropriate in dogs and cats. Three periods of time can be distinguished in the diagnosis of early pregnancy by ultrasound: the earliest phase, where signs of pregnancy can be found in some cases, but accuracy of diagnosis is very low; the succeeding period, where reliable diagnosis is possible, but results are often difficult to achieve and examination can be time consuming; the third phase in which ultrasound diagnosis is very accurate even under practice conditions and can be efficiently carried out on a large scale basis in breeding management. To achieve high accuracy in determining pregnancies a suitable ultrasound scanner with a good image quality and considerable expertise are required. Under field conditions ultrasonic pregnancy diagnosis should be possible: from Day 14 in the horse, from Day 25-28 in cattle, from Day 25 (transrectal), resp. 35 (transcutaneous) in sheep, goats and swine and from Day 24-25 (transcutaneous) in the dog and cat. Before this period of time reliable diagnosis is sometimes possible: between Day 11 and 13 in the horse, between Day 20 and 25 in cattle, between Day 20 and 25 (transrectal), resp. between Day 30 and 35 (transcutaneous) in sheep and goats, between Day 20 and 25 (transrectal) and Day 22 and 35 (transcutaneous) in swine and between Day 19-20 and 24-25 in the dog and cat. Earlier sonographic indications of pregnancy are not sufficiently reliable for large scale accurate pregnancy diagnosis.     

Preliminary characterisation of extracellular serine proteases of Dermatophilus congolensis isolates from cattle,sheep and horses

Dermatophilus congolensis is a filamentous branching actinomycete that causes dermatophilosis, an exudative dermatitis in ruminants. The pathogenesis of this disease is poorly understood and virulence factors of D. congolensis have not been characterised. Culture filtrate (CF) of 14 D. congolensis isolates from cattle, 15 from sheep and four from horses were examined for proteolytic activity using azocasein as a non-specific substrate. The isolates were from a variety of geographical locations. All the isolates examined produced extracellular proteolytic activity. CF from 24 and 48 h cultures and from first and third passages contained proteases. Proteolytic activity was greatest in neutral to alkaline pH (pH 7–10). CF of bovine isolates contained more proteolytic activity than that of ovine isolates. Furthermore, in substrate SDS-PAGE gels containing azocasein the number of proteolytic bands and their molecular weights in CF of bovine, ovine and equine isolates were different, giving distinctive band patterns for isolates from each host species. Three out of four bovine isolates from Antigua gave a fourth band pattern. Bands of equivalent molecular weights to the proteases could not be identified in silver stained SDS-PAGE gels of CF. Serine protease inhibitors had a concentration-dependent inhibitory effect on proteolytic activity in CF and inhibited activity of all proteolytic bands in substrate gels. With the exception of EDTA which had a variable-enhancing effect on activity, inhibitors of other classes of protease had no effect on activity. We conclude that D. congolensis produces a number of extracellular alkaline serine proteases, our results suggest the presence of host-specific variation between isolates and to a lesser extent between isolates from the same host species.     

Studies of electrophoresis on cellulose acetate membrane of serum proteins from normal horses, sheep and pigs

A method for the rapid electrophoresis on a cellulose acetate membrane of serum proteins from horses, sheep and pigs is discussed.The various main globulin fractions in the serum of these animals were experimentally identified.Normal values for the percentage composition of serum from normal horses, sheep and pigs were calculated.In the horse there was great individual variation in the shape of the β-fraction, assumed to be due to different transferrin types. The mean value for β-globulin of 19.5 % in the horse was higher than for the other two species.The albumin percentage was highest in the sheep and lowest in the pig, 48.5 % and 43.2 % respectively. The sheep had the highest γ-globulin percentage, 22.8 %, while the horse had the lowest with 19.0 %.Finally the values were compared with corresponding figures reported by other authors and the results discussed.     

Isolation of Legionella pneumophila from calves and the prevalence of antibodies in cattle, sheep, horses, antelopes, buffaloes and rabbits

The lungs of 139 calves presented for autopsy and 29 healthy slaughtered calves were examined for Legionella by culture and by direct immunofluorescence (DIF) with fluorescein-conjugated antisera. About 17% of the cadaver lungs and 4% of lungs from slaughtered animals were positive by DIF. Legionella organisms were only isolated from the lungs of two cadavers (L. pneumophila, serogroup 1). In a prevalence study of antibodies to Legionella in domestic and wild animals of various species, titers of greater than or equal to 64 were demonstrated by indirect immunofluorescence in sera of 10% of dairy cattle, 5% of beef cattle, 4% of sheep, 22% of antelopes, 35% of horses, 36% of buffaloes and 0% of laboratory rabbits. The isolation of Legionella from lung tissue is evidence for a possible etiologic role of Legionella spp. in natural pathology of animals.     

Leptospiral antibodies in serum from cattle, swine, horses, deer, sheep, and goats: 1973 and 1974.

During 2 years (fiscal years 1973 and 1974), microscopic agglutination tests were performed on 12,565 serums from cattle, swine, horses, deer, sheep, and goats for the detection of leptospiral antibodies. The most frequent presumptive infecting serogroups were Hebdomadis, Pomona, Autumnalis, Ballum, Australis, and Canicola.     

Further studies on the diagnostic value of gamma-glutamyl transpeptidase and 5'-nucleotidase in cattle, sheep and horses

The distribution of 5'-nucleotidase (5'-NT) and gamma-glutamyl transpeptidase (gamma-GT) is similar in the tissues of the sheep, calf and horse, except that there is relatively less gamma-GT in calf liver than in the liver of the other two species. The liver lesion produced by the oral administration of chloroform is similar in the three species and is accompanied by the release of 5'-NT into the plasma of the sheep and calf but not of the horse. Conversely, gamma-GT is released into plasma of the horse but not of the sheep or calf. This difference is not related to the tissue distribution of the two enzymes. The kidney lesion in sheep produced by the intravenous administration of mercuric chloride is accompanied by a reduction in the rate of excretion of an injected dose of inulin and by an increase in the concentration of urea in plasma and in the activity of gamma-GT in plasma and urine. There was no increase in 5'-NT activity in plasma or urine.     

Multiple forms of alkaline phosphatase in horse bone,liver, kidney,duodenum, caecum and serum separated by agarose gel electrophoresis with and without neuraminidase pretreatment

Horse bone, liver, duodenum, caecum and kidney alkaline phosphatases were separated by a commercial agarose gel electrophoresis method with and without neuraminidase pretreatment, following the manufacturer's directions. Tissue extracts were obtained in saline solution and ALP extracted from cell membranes by the butanol method. Electrophoresis was performed using a TRIS/barbital/sodium barbital buffer with detergent, pH 8.6 to 9.0, at 250 V for 30 minutes. Bone, liver and kidney untreated extracts showed two ALP bands each, but with different relative migration (compared to albumin migration). When they were preincubated with neuraminidase, the two bone bands showed a marked decrease in their migration, followed by the kidney ALP bands and the most anodic band of liver Both intestinal untreated extracts showed three bands but with different mobilities. After preincubation with neuraminidase, the three bands of caecum mucosa decreased in their migration, and the most anodic duodenum band disappeared, overlapping the second one. When tissue extracts were incubated with wheat germ-lectin (WGL), 74.5% of bone extract ALP and 67.2% of caecum extract ALP precipitated, which demonstrated that the ALP band of both tissues have similar groups in the carbohydrate side chains. Horse serum showed two electrophoretic bands, which increased to three bands when treated with neuraminidase. ALP from hepatocytes was the dominant isoform, followed by a caecum band. Because the electrophoretic mobilities of some of the tissue bands studied were identical, the neuraminidase agarose electrophoretic method appeared to be a satisfactory alternative to separate them.     

Determination of plasma albumin concentration in healthy and diseased turtles: a comparison of protein electrophoresis and the bromcresol green dye‐binding method

Background: In reptile medicine, plasma chemistry analysis is widely used for the evaluation of an individual's health status. The standard method for the determination of plasma albumin concentration is protein electrophoresis combined with the determination of total protein concentration, but the bromcresol green (BCG) dye‐binding method is also used. The reliability of the BCG method for the measurement of albumin concentration in reptiles is unknown. Objective: The aim of this study was to compare the plasma albumin values of turtles obtained by protein electrophoresis and the BCG method. Methods: Between March 2008 and September 2008, heparinized plasma samples from 16 clinically healthy and 10 diseased turtles of different species were collected. Plasma albumin concentrations were measured by protein electrophoresis and by the BCG method. The results of the 2 methods were compared using Passing–Bablok regression and Bland–Altman plots. Results: Albumin concentration measured by BCG was weakly correlated with the corresponding protein electrophoretic values in all turtles (r_s=.610, P<.001) and in healthy turtles evaluated separately (r_s=.700, P=.003), whereas in diseased turtles no such correlation was found (r_s=.374, P=.287). The albumin concentration measured with the 2 different methods differed significantly in all turtles (P=.009; Wilcoxon's test) and in healthy turtles (P=.005) but not in diseased animals (P=.241). In the Bland–Altman plot a systematic error was found between the 2 methods in diseased turtles. Conclusion: Measurement of albumin by the BCG dye‐binding method may lead to inaccurate results for plasma albumin concentration, especially in ill turtles. Therefore, for health assessment in turtles, albumin should be measured by protein electrophoresis.