Serum protein response and renal failure in canine Babesia annae infection

Babesia annae piroplasms have recently been recognised as a cause of infection and disease among dogs in Europe. The pathogenesis and clinical implications of this emerging disease remain poorly understood. We conducted this study to describe the electrophoretic profiles associated with the infection and to determine if B. annae associated azotaemia is caused by renal failure. We examined by microscopy 2,979 canine blood samples submitted to a diagnostic laboratory in NW Spain between September 2001 and April 2002. Small ring-shaped piroplasms were detected in blood smears of 87 samples and the identity of 58 of these presumptive cases were confirmed by PCR. This group of 58 infected dogs and a reference group of 15 healthy non-infected dogs were our study population. For all the dogs, serum protein response to -albumin, alpha-1 globulin, alpha-2 globulin, beta globulin and gamma globulin- was measured by capillary electrophoresis. The response of infected and non-infected dogs was compared and within infected dogs, the response of those with azotaemia (19) was compared with that of non-azotaemic dogs (39). Infected dogs presented a significant elevation of total proteins and all the different globulin fractions, and significantly lower levels of albumin compared to non-infected dogs. Among infected dogs, those presenting azotaemia had significantly lower concentrations of total proteins, albumin, beta and gamma globulins, and significantly higher values of alpha-2 globulin. Specific gravity was below the threshold of 1,025 for all dogs with azotaemia for which a urine sample was available (7) suggesting that azotaemia, in these dogs was of renal origin. Azotaemic dogs had higher concentrations of cholesterol and triglycerides, probably as a result of a liver compensatory response to the loss of proteins. We conclude that serum protein response in B. annae infected dogs corresponds to the pattern of a haemolytic syndrome with intense inflammatory reaction and that the azotaemia associated to the infection is very likely of renal origin.     

Serum proteins in normal cats and cats infected with Aelurostrongylus abstrusus

Serum proteins were evaluated by cellulose acetate electrophoresis in cats prior to and every 2 weeks for 24 weeks after oral infection with third stage larvae of Aelurostrongylus abstrusus. Evaluation of electrophoretograms was standardized by determination of electrophoretic migration ratios. Six fractions of serum proteins were consistently identified: albumin and alpha 1, alpha 2, beta 1, beta 2, and gamma globulin. Relative and absolute concentrations of each serum protein fraction were determined. The only changes found were a decrease in concentration of alpha globulins and an increase in concentration of beta 1 globulins. These changes were mild, however, so that the the concentrations for infected cats were still within 1 SD of the control concentrations. Apparently serum electrophoresis is not a useful diagnostic test for aelurostrongylosis.     

Agarose gel serum protein electrophoresis in cats with and without lymphoma and preliminary results of tandem mass fingerprinting analysis

Background: Serum electrophoretic profiles in cats are poorly characterized with respect to the proteins that comprise the globulin fractions, and interpretation of the electrophoretograms is routinely done in the absence of information about identity of the proteins found within each fraction. Objectives: The aims of this study were to compare protein fractions separated by serum protein electrophoresis (SPE) in healthy cats and in cats with lymphoma and to confirm some component proteins in the major fractions following SPE using tandem mass fingerprinting analysis (TMFA). Methods: Total protein concentration was measured and agarose gel SPE performed on serum from 14 healthy cats and 14 cats with lymphoma. The absolute protein concentration within each fraction was compared between the 2 groups. Bands corresponding to the SPE fractions were excised from the gels of 2 control cats and 1 cat with lymphoma and analyzed by liquid chromatography coupled to mass spectrometry. Results were compared with sequences in the National Center for Biotechnology Information protein database. Results: Median albumin concentrations were significantly decreased and median β‐globulin concentrations were significantly increased in cats with lymphoma. Narrow electrophoretic spikes were present in the β/γ‐globulin fraction in 3 cats with lymphoma. Following TMFA, multiple proteins were identified in each fraction, and their mobility agreed with results from previous studies generated using alternative techniques. Inter‐α (globulin) inhibitor 4 was identified in feline serum for the first time. Conclusions: Cats with lymphoma had lower albumin and higher β‐globulin concentrations than did healthy cats. Despite limitations of one‐dimensional agarose gel SPE, TMFA provided preliminary data to confirm the protein components of the various fractions.     

Electrophoretic studies on serum proteins in cows with traumatic pericarditis.

Diagnostic significance of electrophoretic findings of serum protein in cows with traumatic pericarditis was evaluated. Affected cows were classified into 3 groups according to autoptical findings: fibrinous, sero-fibrinous, and purulent types. Slight hypoprotenemia, moderate hypo-albuminemia, slight hyper-alpha globulinemia and tendency of hyper-beta globulinemia were commonly observed in the affected cows. The level of gamma globulin tended to be lower in the cows with fibrinous or sero-fibrinous, and higher in purulent pericarditis, than the level in healthy cows. In the serum protein electropherograms of the cows with fibrinous or sero-fibrinous pericarditis, there was pathognostic pattern composed of slender albumin, acute shape of alpha globulin with a broad rising accompanied by double peaks and with main peak migrating toward the albumin side, tendency of rising beta globulin fraction, and large indentation between beta and gamma fractions. These findings except for the slender albumin fraction, however, was not or poorly observed in purulent pericarditis. Electrophoretic findings were subacute inflammatory pattern with non-selective serum protein losing in fibrinous or sero-fibrinous, and chronic inflammatory pattern in purulent pericarditis.     

Electrophoretic and immunoelectrophoretic analysis of feline serum proteins.

Serum from 28 clinically healthy cats was subjected to agarose gel electrophoresis and the migration distance relative to albumin was determined. The reference values for the relative and absolute concentrations of each protein fraction were determined and compared to previous reports. The immunoelectrophoretic, crossed immunoelectrophoretic and crossed line immunoelectrophoretic pattern, of a pooled sample of serum from clinically normal cats was determined. The cross-reactivity between goat and/or rabbit monospecific antisera to human proteins and feline serum was determined using immunoelectrophoresis and crossed-line absorption immunoelectrophoresis. Feline alpha-2-macroglobulin, haptoglobin, B1C-globulin, IgG, albumin and ceruloplasmin cross reacted strongly with the monospecific antisera. Alpha-2-macroglobulin migrated anodal to haptoglobin. Lipoproteins and ceruloplasmin were studied using staining procedures described in man. Feline transferrin was precipitated with Rivanol.     

Canine serum protein patterns using high-resolution electrophoresis (HRE)

Serum protein values were determined in 26 healthy dogs using agarose gel electrophoresis (SPE), splitting the electrophoretic separation into six regions: albumin, alpha(1), alpha(2), beta(1), beta(2)and gamma globulins. High-resolution electrophoresis (HRE) was used to separate single proteins. Serum proteins from dogs (26 healthy and 20 affected by various diseases) were then characterized by electrophoretic immunofixation (IFE) and Sudan black staining on HRE film. Haemoglobin and normal canine plasma and serum were used to identify haptoglobin and fibrinogen, respectively.In the standard pattern, determined by HRE, the following proteins were identified: albumin, alpha(1)-lipoprotein (alpha(1)-region), haptoglobin and alpha(2)-macroglobulin (alpha(2)-region), beta -lipoprotein and C3 (beta(1)-region), transferrin and IgM (beta(2)-region), IgG (mostly in gamma -region and partly in beta(2)-region). The HRE pattern shown by healthy dogs could be compared with those of dogs affected by various diseases to obtain clinical information.     

Analysis of serum proteins in clinically normal pet and colony cats, using agarose electrophoresis.

The relative and absolute values of the electrophoretic fractions of serum proteins of 50 clinically normal cats were determined, using agarose as the supporting matrix. Six protein fractions were clearly and consistently resolved: albumin and alpha1-, alpha2-, beta1-, beta2-, and gamma-globulins. In many cats, the alpha1-, alpha2-, and beta2-fractions were each divided into 2 subfractions. Cats which lived in a research colony environment were found to have significantly increased levels of gamma-globulins as compared with the values in cats kept as house pets. The results of serum protein fractionation using this technique have been compared with the normal feline values reported in the literature.     

Lipids and lipoproteins in normal dogs and in dogs with secondary hyperlipoproteinemia.

Concentrations of serum cholesterol, triglyceride, and free glycerol were neasured, and serum lipoprotein electrophoresis was performed in normal dogs and in dogs with hypothyroidism, diabetes mellitus, and acute pancreatitis. Twelve privately owned dogs and 20 Basset Hounds from a research colony served as normal subjects. Privately owned had higher serum lipid concentrations than did research dogs. On electrophorisis of normal dog serum, lipoprotein bands were detected at the beta, pre-beta, alpha-2, and alpha-1 positions. Hypercholesterolemia was associated with increased intensity of the alpha-2-lipoprotein band, and hypertriglyceridemia occurred with increased lipoprotein at the origin, or the beta or pre-beta positions. Hypothyroid dogs had normal lipid values and lipoprotein electrophoretic patterns, hypercholesterolemia with increased intensity of the alpha-2-lipoprotein band, or hypercholesterolemia and hypertiglyceridemia with prominent beta-, pre-beta, and alpha-2-lipoprotein electrophoretic patterns were changed to near-normal values after levothyroxine administration. Diabetic dogs had increased serum cholesterol and triglyceride content and a predominance of lower density lipoproteins, as detected by electrophoresis. Insulin therapy caused the lipid concentrations to decrease and the electrophoretic pattern to revert to near normal. Dogs with acute pancreatitis had moderately increased serum lipid content and electrophoretic patterns characterized by increased intensity of the beta-lipoprotein band and by altered migration of alpha-1-lipoproteins.     

Serum protein reference values in foals during the first year of life: comparison of chemical and electrophoretic methods

Normal reference values for serum proteins of foals from birth to 1 year of age have been established. Chemical and electrophoretic/refractometric methods for total protein, albumin, total globulin and Albumin/Globulin (A/G ratio) have also been compared. The biuret total protein method and Bromcresol Green (BCG) method on the Technicon SMA 12/60 autoanalyzer were used and compared with total protein determined via refractometry and albumin determined by Cellulose Acetate (CA) electrophoresis/densitometry. Globulin and A/G ratios were calculated from the chemical method data and compared with that obtained electrophoretically. Total protein, albumin, total globulins and A/G ratios all were in agreement at all sample times studied. Data on the subfractionation of serum globulins via CA electrophoresis is also presented. Wide variations in alpha and beta globulin levels were noted among the foal sera early in life. As a result, two distinct populations of foals with respect to both globulin content and A/G ratio were identified. One of these populations (Group A) appeared to have obtained passive immunity more slowly than the other (Group B) animals. Comparison of these data with clinical cases of foals in which failure of passive transfer was a part suggests that the A/G ratio may be useful in assessing adequate colostral antibody levels in the newborn foal.     

Serum protein electrophoresis in spontaneous canine hyperadrenocorticalism

The serum protein concentrations of dogs with confirmed spontaneous hyperadrenocorticalism were determined by agarose gel electrophoresis before and during treatment with mitotane. In untreated animals a significant increase was detected in the mean concentration of total protein and the mean concentration and percentage of alpha-2 globulin. The mean concentration and percentage of albumin and gamma-globulin were significantly decreased. In animals on treatment the mean concentration of total proteins and the mean concentration and percentage of beta-2 globulin were significantly reduced.     

Electrophoretic and Chemical Studies on Sera of Swine Following the Feeding of Toxic Groundnut Meal

The consumption of toxic groundnut meal by swine leads to marked alterations in their electrophoretic serum patterns. Experiments carried out by chemical assays and by the moving boundary electrophoretic method of Tiselius and Smithies' starch gel electrophoresis showed a relative decrease in the albumin, alpha₁-, alpha₂-, and beta globulins, while gamma globulin appeared to be considerably increased.     

Studies on the synovia in healthy horses with particular reference to the protein composition.

Synovial fluid and blood were collected from 18 clinically healthy brood mares in resting conidition. The following parameters were analysed: total leucocytes, glucose, alkaline phosphatase (AP), lactate dehydrogenase (LDH), total protein, albumin, total globulin, albumin/globulin ratio and electrophoretic protein picture. The serum/synovia ratios were calculated for all parameters. It was considered to be of greater diagnostic value to compare these serum/synovia ratios rather than to look at the individual concentrations in synovia. The results obtained did not materially differ from those in the existing literature. In addition, this study confirmed that small protein molecules could more easily penetrate the synovial membrane than high-molecular proteins.     

Comparative albumin determinations in ducks, chickens, and turkeys by electrophoretic and dye-binding methods

Serum total protein, albumin, and globulin concentrations in male ducks, turkeys, and chickens were compared, using electrophoretic and dye-binding methods, as well as using a bovine and chicken albumin standard. When a chicken standard was used for determination of albumin and globulin concentrations by automated methods, results were more comparable with results of electrophoresis than were those when a bovine standard was used.     

Liver abscesses in intensively fed cattle. Serial investigations of serum proteins

An attempt was made to determine the time of onset of liver abscesses in intensively fed calves. Determinations of seram proteins (total protein, albumin, globulin, albumin/ globulin ratio, agarose-gel electrophoretic separation and formol-gel test) and, in a part of the material, of serum-GOT and -GPT were made in serial samples taken once a month from 125 calves. Liver abscesses were found in 48 (38.4 %) when slaughtered at 6–7 months of age.Total protein and globulin increased, the albumin-globulin ratio decreased with increasing age. Calves with liver abscesses had a significantly (0.05 > P > 0.01) higher globulin concentration at the last sampling. At this sampling the material was of a limited size and the difference has to be interpreted with caution. No other significant differences were found. Nor were any rises observed in S-GOT and S-GPT referable to the formation of liver abscesses.Bacteriological studies were not made but, in analogy with observations on a similar animal material in another part of Sweden, the infectious agent in the liver abscesses was assumed to be Sph. necrophorus. The explanation of the almost absent changes in the serum proteins in calves with liver abscesses may be the low antigenicity of this bacterium.In conclusion it is stated that the present serial investigations of the serum protein pattern in young cattle provide no definite guidance for establishing the time of onset of liver abscesses.     

Effects of haemolysis,lipaemia, bilirubinaemia and fibrinogen on protein electropherogram of canine samples analysed by capillary zone electrophoresis

The possible interference of haemolysis, lipaemia, bilirubinaemia and fibrinogen on capillary zone electrophoresis of canine samples were studied. Solutions of haemoglobin, lipid and bilirubin were prepared and mixed with serum aliquots to make up samples containing different concentrations of the putative interferent substance. In addition, samples of serum and plasma were assayed to assess the influence of fibrinogen. Haemolysis and lipids produced a change in electropherogram morphology giving an interference peak located in the beta-2 region when haemoglobin was increased, and in the alpha-2 region when lipids were increased. A rise in concentration of these interferents caused an increase in the beta and alpha-2 fractions respectively, and a decrease in the other fractions. Bilirubin did not alter morphology but gave an increase in the albumin and alpha-1 and a decrease in the alpha-2 and beta-2 fractions. No differences were found between serum and plasma samples, and fibrinogen did not produce any additional peak.     

Analysis of serum proteins, using agarose electrophoresis in normal dogs and in dogs naturally infected with Dirofilaria immitis.

The electrophoretic pattern of 130 serum samples from clinically normal dogs was evaluated, using agarose as the supporting matrix. The relative mobility of each globulin fraction in relationship to the mobility of albumin was determined in 70 dogs, and nomenclature based on the mobilities (Rf values) was proposed. Biuret protein determinations were done, and relative and absolute values of each serum protein fraction were determined. Changes in the fractions of serum proteins were evaluated in regard to sex and age of the dogs and the presence of microfilariae of Dirofilaria immitis, as determined by modified Knott tests.     

Determination of serum albumin in domestic animals using the immediate bromeresol green reaction

The immediate reaction bromeresol green (BCG) method for determination of serum albumin in various species of domestic animals (bovine, equine, canine, feline and porcine) was compared to an electrophoretic method. Good correlation was obtained between the two methods when both species specific and bovine albumin standards were used to compute the albumin concentration for the BCG assay. The immediate reaction BCG method appears to have considerable potential for application in the veterinary clinical laboratory.     

A preliminary study on the serum protein response in canine babesiosis

Total serum protein, albumin, globulin, globulin fractions (alpha, beta and gamma globulins) and an acute-phase protein (alpha1-acid glycoprotein) were evaluated in dogs with naturally occurring mild (Group 1), severe (Group 2) or complicated babesiosis (Group 3). Results showed that the total serum protein, albumin, A/G ratio and alpha globulins were statistically different between Groups 1 and 2. There was no statistical difference between groups with total, beta and gamma globulins. The findings from this study suggest that dogs with mild and severe babesiosis had low total serum proteins, albumin, A/G ratio and alpha globulins; dogs with complicated babesiosis showed no typical serum protein changes or patterns; and that there was no evidence of an acute-phase response detectable on serum protein electrophoresis in any of the 3 groups. A marked acute-phase response was, however, present, as measured by the alpha1-acid glycoprotein, in all 3 groups. As this was a retrospective study, the possibility that the observed responses were due in part to concurrent disease could not be excluded.     

Electrophoretic serum protein fraction profile during the different physiological phases in Comisana ewes

The aim of this study was to evaluate the influence of different physiological phases on serum total proteins and their fractions of ten Comisana ewes housed in Mediterranean area. From each animal, blood samples were collected at different physiological phases: late pregnancy, post-partum, early, mid-, end lactation and dry period. On all samples serum total proteins were determined by the biuret method, and albumin, α-globulins, β(1) -globulins, β(2) -globulins and γ-globulins concentrations were assessed using an automated system. One-way repeated measures analysis of variance was applied to determine the significant effect of different physiological phases on the parameters studied. During the late pregnancy and post-partum, total proteins, β1- and β2-globulins and γ-globulins showed the highest values. Starting from post-partum, α-globulins increased to reach their peaks in mid-lactation. Early lactation was characterized by low γ-globulins values. The increase in serum albumin concentration and the drop in some globulin fractions determined the significant increase in albumin/globulin ratio. The obtained results contributed to improve the knowledge on electrophoretic profile during the different physiological phases in ewes, confirming that pregnancy and lactation periods affect the protein metabolism. Particularly, serum protein fractions pattern could give information about dehydration, plasma volume expansion and hepatic function, which occur during the different physiological phases. Dynamics of the protein profile - from pregnancy to dry period - which are provided by our results, could be considered as guidelines for the management strategies to guarantee the nutritional needs of these animals during the different physiological phases and to avoid a decline of productive performance and consequently an economic loss.     

Alkaline phosphatase isoenzymes in feline serum using an agarose gel alkaline phosphatase kit method.

Total serum alkaline phosphatase (ALP) activity is the product of the combined activity of isoenzymes from a number of tissue sources. In this study, a commercially available kit for electrophoretic separation of ALP isoenzymes in an agarose gel was used to separate ALP isoenzymes in feline tissue extracts and serum. Five separate bands of ALP activity were identified. These bands were numbered 1 to 5 with band 1 having the most anodal migration. The tissue of origin corresponding to the migration position of the isoenzymes are as follows: Band 3 was the liver isoenzyme, band 4 was the bone isoenzyme and ALP isoenzymes of both intestine and kidney migrate in the position labelled band 5. Band 1 appears to be related to albumin and does not represent true ALP activity. The tissue source of band 2 (a and b) was not identified. Serum ALP activity of mature, healthy cats is primarily of liver origin. Immature cats (< 1 year of age) have a greater proportion of the bone isoenzyme in the serum.