The fibrinogen concentration in blood of dairy cows and its influence on the interpretation of the glutaraldehyde and formol-gel test reactions.

It has earlier been shown that the formol-gel test on serum and glutaraldehyde test on whole blood are simple and rapid methods for evaluation or the immunoglobulin status in the cow. Both tests function as coagulation tests in which aldehyde groups oross-link basic blood globulins at their NH₂-groups, forming polymerisates. The glutaraldehyde has in whole blood the capacity to polymerize not only immunoglobulins but also fibrinogen.This investigation was made in order to study whether the fibrinogen level may influence the result of the glutaraldehyde test, so revealing any differences between the results of that and the formol-gel test carried out on serum. In 92 cows with a variety of clinical disorders (most of them with inflammatory processes) the total protein, albumin, total globulin concentration and albumin/globulin ratio in serum and fibrinogen concentration in plasma were recorded. The material was grouped according to glutaraldehyde and formol-gel test reactions.It is shown that increases in the fibrinogen level have an effect on the results of the glutaraldehyde test. A positive glutaraldehyde test in more acute processes is ascribed to a heavy rise of plasma fibrinogen in its capacity of acute-phase protein. A positive glutaraldehyde test in chronic diseases may be viewed as a result of interaction between high immunoglobulin concentrations and elevated fibrinogen concentration.In conclusion the fibrinogen and immunoglobulin status of blood is important to assess in many diseases of cattle. The semiquantitative tests described for field use can separately, or especially in parallel use, provide valuable information about the character and development of a disease and may be regarded as good substitutes for the sedimentation rate (SR), which is not demonstrable in cattle. kw|Keywords|k]bovine fibrinogen; k]bovine serum proteins; k]formol-gel reaction; k]glutaraldehyde test; k]acute and chronic inflammations     

Studies on Diarrhea in Neonatal Calves: The Plasma Proteins of Normal and Diarrheic Calves During the First Ten Days of Age

The concentration of serum proteins and plasma fibrinogen were determined in 151 normal and 49 diarrheic calves at intervals from birth to ten days of age. There were significant differences in the concentrations of the various serum proteins in normal calves when the results were analysed at six age intervals. There was no significant relationship between the concentration of the various proteins and the season of the year.

Of the diarrheic calves, those that died had significantly lower gamma globulin concentrations than the other calves. Severely diarrheic and dehydrated calves had significantly increased serum albumin and alpha glabulin concentrations and decreased gamma globulin concentrations. No significant variation occurred in total serum protein concentration. Plasma fibrinogen concentrations were similar in normal and diarrheic calves.

     

An investigation of the ability of the glutaraldehyde test to distinguish between acute and chronic inflammatory disease in horses

The glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

The Value of the Glutaraldehyde and Formaldehyde Tests in Evaluation of the Globulin Level in Bovine Blood

So-called liability reactions are based on the gelification of blood globulins when reacting with, i.e., glutaraldehyde or formaldehyde. Solidifying time is inversely proportional to the globulin concentration of the blood. In the present investigation glutaraldehyde and formaline-gelification tests were compared concerning their diagnostic and prognostic value in dairy cows with internal diseases.The investigation confirmed earlier results. The correlation between globulin concentration and gelification time was significant, and both tests give very good possibilities to reveal hyper-γ-globulinaemias.The 2 tests were relatively equal as regards the reliability. Even though the formol-gel reaction allows a somewhat better safety and semi-quantitative differentiation than the glutaraldehyde test, the latter has obvious practical advantages. It can be done on whole blood and with a short observation time. Thus, the blood globulin status can be evaluated during the visit on the farm.     

Comparison of rapid laboratory tests for failure of passive transfer in the bovine

Background

Failure of passive transfer of maternal immunity via colostrum can occur in the bovine, and a number of blood tests have been developed to test calves for this failure. It is not clear which test is most suitable for this purpose. The objective was to examine the most commonly used tests for failure of passive transfer and to decide which is most suitable for routine laboratory use. 126 serum samples were taken from calves of dairy cows after birth but prior to colostrum feeding, and at 48 h of age. Five different tests were compared against radial immunodiffusion which is considered the appropriate reference method. These tests were serum gamma-glutamyltransferase levels, serum protein levels, serum globulin levels, an enzyme linked immunosorbent assay and the zinc sulphate turbidity test.

Results

The tests examined displayed high sensitivity but widely varying specificity. Examination of the use of different cut-off points allowed some improvement in specificity at the expense of sensitivity, but the tests which had performed best at the original cut-off points still displayed the best performance. Gamma-glutamyltransferase levels as a measure of colostrum absorption returned, in this study, the best balance between sensitivity and specificity. The ELISA used in this study and serum globulin levels displayed performance similar to the gamma-glutamyltransferase levels. Serum total protein was less successful than others examined at providing both sensitivity and specificity but may, when performed via refractometer, be useful for on-farm testing. As currently performed the poor sensitivity for which the zinc sulphate turbidity test is most often criticized is evident. Modification of the cut-off point to increase specificity is less successful at balancing these parameters than the ELISA, gamma-glutamyltransferase levels, and globulin levels.

Conclusions

Gamma-glutamyltransferase levels, ELISA testing and circulating globulin levels performed best in detecting failure of passive transfer in serum samples, although all three had some practical considerations.     

Comparison of methods used to diagnose generalized inflammatory disease in manatees (Trichechus manatus latirostris).

Manatees (Trichechus manatus latirostris) are afflicted with inflammatory and infectious disease secondary to human interaction, such as boat strike and entanglement, as well as "cold stress syndrome" and pneumonia. White-blood-cell count and fever, primary indicators of systemic inflammation in most species, are insensitive in diagnosing inflammatory disease in manatees. Acute phase-response proteins, such as haptoglobin and serum amyloid A, have proven to be sensitive measures of inflammation/infection in domestic large animal species. This study assessed diagnosis of generalized inflammatory disease by different methods including total white-blood-cell count, albumin: globulin ratio, gel electrophoresis analysis, C-reactive protein, alpha, acid glycoprotein, haptoglobin, fibrinogen, and serum amyloid A. Samples were collected from 71 apparently healthy and 27 diseased animals during diagnostic medical examination. Serum amyloid A, measured by ELISA, followed by albumin:globulin ratio, measured by plasma gel electrophoresis, were most sensitive in diagnosing inflammatory disease, with diagnostic sensitivity and specificity of approximately 90%. The reference interval for serum amyloid A is <10-50 microg/ml with an equivocal interval of 51-70 microg/ml. The reference interval for albumin:globulin ratio by plasma gel electrophoresis is 0.7-1.1. Albumin: globulin ratio, calculated using biochemical techniques, was not accurate due to overestimation of albumin by bromcresol green dye-binding methodology. Albumin:globulin ratio, measured by serum gel electrophoresis, has a low sensitivity of 15% due to the lack of fibrinogen in the sample. Haptoglobin, measured by hemoglobin titration, had a reference interval of 0.4-2.4 mg/ml, a diagnostic sensitivity of 60%, and a diagnostic specificity of 93%. The haptoglobin assay is significantly affected by hemolysis. Fibrinogen, measured by heat precipitation, has a reference interval of 100-400 mg/dl, a diagnostic sensitivity of 40%, and a diagnostic specificity of 95%.     

Evaluation of white blood cell concentration,plasma fibrinogen concentration,and an agar gel immunodiffusion test for early identification of foals with Rhodococcus equi pneumonia

OBJECTIVE: To evaluate WBC concentration, plasma fibrinogen concentration, and an agar gel immunodiffusion (AGID) test for early identification of Rhodococcus equi-infected foals. DESIGN: Prospective study. ANIMALS: 162 foals from a farm with enzootic R equi infection. PROCEDURE: Blood samples were obtained from each foal at 4-week intervals for measurement of WBC and plasma fibrinogen concentrations and at 2-week intervals for detection of anti-R equi antibody by an AGID assay. Diagnostic performance of WBC and fibrinogen concentrations was assessed by use of receiver operating characteristic curve analysis. For each assay, sensitivity, specificity, and predictive values were calculated at various cutoff points; bacteriologic culture of R equi from a tracheobronchial aspirate was used as the reference standard test. RESULTS: Diagnostic performance of WBC concentration was significantly higher than that of fibrinogen concentration. Sensitivity and specificity of measurement of WBC concentration at a cutoff of 13,000 cells/microL were 95.2 and 61.2%, respectively; at a cutoff of 15,000 cells/microL, sensitivity was 78.6% and specificity was 90.8%. When a positive test result was used as the cutoff, sensitivity of the AGID assay was 62.5% and specificity was 53.8%. CONCLUSION AND CLINICAL RELEVANCE: Monitoring WBC concentration is a useful approach for early detection of infected foals on farms with a high prevalence of R equi pneumonia. In contrast, serologic surveillance by use of an AGID assay is of little benefit for that purpose.     

Glutaraldehyde test on whole blood of swine at slaughter--a preliminary report]

In an initial experiment the highest concentration of a glutaraldehyde solution that does not coagulate whole blood of fatteners without or with only slight pathological changes was established. Total protein, albumin, globulin, A/G ratio and electrophoretic protein pattern were at the same time determined in blood serum. A glutaraldehyde solution with the established highest concentration was used in a second experiment on whole blood to distinguish fatteners without or with only small pathological changes from those with severe processes such as abscesses and purulent arthritis. It is evident that the glutaraldehyde test gives a good idea of the immunoglobulin status of fatteners. There is reason to believe that systematized glutaraldehyde testing of whole blood taken at pig slaughter could improve the chances of detecting concealed inflammatory processes.     

Comparison of in vitro tests for evaluation of passive transfer of immunoglobulins in giraffe (Giraffa camelopardalis).

Serum samples from captive giraffe (Giraffa camelopardalis) were tested to assess passive transfer of immunoglobulins using in vitro methods developed for domestic ruminants. Estimated immunoglobulin levels were compared using five tests (protein electrophoresis, total protein refractometry, zinc sulfate turbidity, glutaraldehyde coagulation, and sodium sulfite turbidity). A linear relationship was observed among total protein, gamma globulin (electrophoretic measurement), and immunoglobulin level based on spectrophotometric measurement of zinc sulfate turbidity. Nonquantitative assays also demonstrated statistical correlation with the quantitative methods. Using criteria similar to those established for domestic species, cutoff values for failure of passive transfer (FPT) were established for these tests in neonatal giraffe: 1) total protein <6.0 g/dl; 2) gamma globulin < 0.5 g/dl; 3) estimated immunoglobulin level < 1,000 mg/dl (zinc sulfate turbidity); 4) glutaraldehyde coagulation test negative; or 5) no visually detectable turbidity in 16% sodium sulfite or Bova-S negative. Retrospective examination of the medical histories showed a strong statistical association between animals designated as having FPT and those that were removed from their dams based on clinical assessment to be hand-reared. Application of these tests in the field should allow earlier detection and intervention for FPT in neonatal giraffe.     

Changes in the blood biochemical and haematological profile of neonatal calves with age

Fourteen calves were used to investigate the changes from birth to 83 days of age in the concentrations of serum albumin, alkaline phosphatase, beta-hydroxybutyrate, plasma cortisol, serum creatine kinase, creatinine, iron, plasma fibrinogen, serum gamma-glutamyl transferase, plasma glucose, haptoglobin, serum non-esterified fatty acids, total protein, transferrin, triglycerides, urea and gamma globulin; the haematological variables measured were: basophils, eosinophils, haematocrit, haemoglobin, lymphocytes, mean cell haemoglobin, mean cell haemoglobin concentration, mean cell volume, monocytes, band neutrophils, neutrophils, platelets, red blood cells and white blood cells. The changes are presented as a series of graphs and the values are discussed in relation to the published reference ranges for adult cattle. Two populations of calves were identified which gave rise to a bimodal distribution for some of the variables. Differences in haematocrit, haemoglobin and red blood cell counts were apparent at birth, with raised values for these measurements being associated with an increased white blood cell and neutrophil count between three and 27 days of age.     

Fluorescence polarization assay for the diagnosis of bovine brucellosis: adaptation to field use

A fluorescence polarization assay (FPA) was used to test whole blood samples prepared by mixing blood cells from cattle without exposure to Brucella abortus (B. abortus) with sera from animals with confirmed (bacteriologically) infection. A cut-off value between negative and positive values was initially established to be 87.2mP. This value was changed to 95mP to increase assay specificity without loss of sensitivity when testing blood samples from negative animals. The FPA technology was applied to whole blood samples in the field and to stored whole blood samples using two diluent buffers. Relative sensitivity and specificity values for the FPA performed in the field, based on buffered antigen plate agglutination test and competitive enzyme immunoassay results were 95.3 and 97.3%, respectively. However, to obtain maximum sensitivity and specificity, a cut-off value of 105mP was determined for fresh whole blood samples. The relative sensitivity and specificity values of the FPA when testing stored whole blood samples were 100% each using a 95mP cut-off.The usefulness of the FPA for testing whole blood samples in the field was demonstrated.     

Effect of immunization on the quantitative behavior of gamma globulins in the blood serum of cattle following whole-body x-ray irradiation

Five experiment series were applied to 38 calves, aged between two-and-a-half and four months, using whole-body exposure to X-ray treatment based on median lethal doses (MLD) between 1.2 Gy and 1.7 Gy. Various methods of immunisation were simultaneously applied, and their effects on irradiation-caused response of gamma globulin concentrations in blood serum were studied. Gamma globulin levels were determined by Kunkel's zinc sulphate turbidity test. Immunisation applied between 14 and 21 days prior to irradiation resulted on obvious stimulation of gamma globulin production. In two experiments with parenteral antigen application, gamma globulin levels increased through about 14 days in apparently irradiated animals declined, after that period of time, but they continued to rise in really irradiated probands. A high lethal dose of 1.7 Gy, however, was followed by significant drop, three weeks after irradiation, that is in concomitance with the climax of the radiation syndrome. When 1.5 Gy were used, rise in gamma globulin concentration was recordable also following oral administration of antigen. Response of irradiated animals was even more clearly pronounced by secondary reaction of antibody formation, following booster action due to experimental infection with homologous germs. The stimulating effect of irradiation upon gamma globulin levels in blood serum, when oral or parenteral immunisation had taken place, prior to irradiation, has been attributed to reaction of the immune-globulin producing system to the release of tissue proteins or tissue antigens. Immunisation, prior to irradiation, stimulated the immunological system which underwent stronger regeneration, after irradiation, or had obviously acquired higher resistance to radiation.     

Long term haematological and serum protein changes following splenectomy in sheep

Splenectomy was performed on five rams since four years to study the long-term effect of the operation on blood profile and serum total proteins and paper electrophoresis.A reduction in the blood profile was accompanied with a decrease in the betta, gamma globulin and total globulin following removal of the spleen. Alpha globulin showed a slight rise. The albumin/globulin ratio was highly increased.     

Effects of hyperosmolar ionic and low osmolar non-ionic contrast media on coagulation times and some blood parameters in dogs: an in vivo study

The purpose of this study is to evaluate the effects of hyperosmolar ionic contrast media (CM) (diatrizoate) and low osmolar non-ionic CM (iohexol and ioxilan) on coagulation time and some blood parameters in dogs in vivo. The animals were divided into three groups in equal numbers. The dogs in groups I, II and III received diatrizoate, iohexol and ioxilan at the dose of 700 mgI/kg intravenously (IV) as a bolus, respectively. Administration of contrast media and blood samples were collected from vena cephalica antebrachii prior to CM administration and thereafter at 3, 15, 30, 60, 90 and 180 min and 24 h to measure the coagulation factors [activated partial thromboplastin time (APTT), prothorombin time (PT), fibrinogen and fibrinogen degradation products] and some other blood parameters [red blood cells, platelet, white blood cells, haematocrit (Ht) and haemoglobin (Hb)]. While a statistically significant decrease was observed on APTT at 15 min in group III, no significant differences were found in groups I and II. All the groups had insignificant alterations for PT, fibrinogen and fibrinogen degradation product, following CM administration. Significant decreases were observed for platelet at 3 min in all groups. This decrease was also significant at 15- and 30- min intervals in group I. There were significant decreases for erythrocytes, Ht and Hb measurements within 30 min, and no significant alterations were observed for leucocytes within 60 min in all groups compared with baseline values. No differences were observed with regard to coagulation times and some blood parameters as far as long-lasting and major effects of each CM are concerned.     

Simulation of genetic control of reproduction in beef cows. I. Simulation model

A stochastic computer simulation model was constructed to predict the reproductive performance of beef cows as a function of the postpartum interval from calving to first estrus and the single-service conception rate. Phenotypic values for single-service conception rate were obtained by truncation of an assumed underlying normal phenotypic distribution at a point corresponding to a cumulative frequency of conception of .70. The underlying phenotypic distribution was derived as the sum of independent, normally-distributed additive genetic, permanent environmental and temporary environmental effects. The phenotypic frequency distribution for postpartum interval has been shown to be skewed toward large intervals. This distribution was simulated by combining normal distributions of additive genetic and permanent environmental effects with a strongly skewed Pearson III gamma distribution of temporary environmental effects. The simulated management system involved a 63-d breeding season, thereby allowing females up to three opportunities to conceive. The reproductive outputs that were simulated from postpartum interval and single-service conception rate included date of first service, first-service conception rate, number of services received, annual conception rate, calving date and postpartum interval. A normally-distributed 205-d weaning weight was also simulated and converted to an actual weaning weight by adjustment for calving date. General simulation results in terms of frequencies, means and standard deviations were compared with values found in the literature and found to provide a reasonable model of bovine reproductive performance.     

Approaches towards optimising the gamma interferon assay for diagnosing Mycobacterium bovis infection in African buffalo (Syncerus caffer)

The application of diagnostic tests for bovine tuberculosis in wildlife poses formidable technical difficulties and the use of the gamma interferon assay offers a simplified approach to testing wild animal species. We compared the performance of the gamma interferon assay in African buffalo (Syncerus caffer) under the recommended guidelines for interpretation of test results and found a high sensitivity (92.1%) at the cost of a greatly reduced specificity (68.3%). The optimised cut-off value for positive test results under local conditions was identified at an optical density of 0.385 at wavelength 450nm as the preferred compromise between sensitivity and specificity. Additional optimisation approaches to improve test performance were examined and showed that the application of 'a priori exclusions' of test results on the basis of reactivity to fortuitum PPD (sensitin produced from Mycobacterium fortuitum) and to a lesser degree, avian PPD, increased specificity without losing sensitivity. The implications of these findings on a modified testing protocol adjusted to include measurement of immune responsiveness to fortuitum PPD and other interpretation schemes are discussed.     

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

We review the principles and practical application of receiver-operating characteristic (ROC) analysis for diagnostic tests. ROC analysis can be used for diagnostic tests with outcomes measured on ordinal, interval or ratio scales. The dependence of the diagnostic sensitivity and specificity on the selected cut-off value must be considered for a full test evaluation and for test comparison. All possible combinations of sensitivity and specificity that can be achieved by changing the test's cut-off value can be summarised using a single parameter; the area under the ROC curve. The ROC technique can also be used to optimise cut-off values with regard to a given prevalence in the target population and cost ratio of false-positive and false-negative results. However, plots of optimisation parameters against the selected cut-off value provide a more-direct method for cut-off selection. Candidates for such optimisation parameters are linear combinations of sensitivity and specificity (with weights selected to reflect the decision-making situation), odds ratio, chance-corrected measures of association (e. g. kappa) and likelihood ratios. We discuss some recent developments in ROC analysis, including meta-analysis of diagnostic tests, correlated ROC curves (paired-sample design) and chance- and prevalence-corrected ROC curves.     

Use of ESAT-6 in the interferon-gamma test for diagnosis of bovine tuberculosis following skin testing

The whole blood interferon-gamma (IFN-gamma) test has proven to be a practical ancillary test for re-testing cattle for bovine tuberculosis 8-28 days following tuberculin skin testing. An improvement in the specificity of the IFN-gamma test could further reduce culling of false positive animals. The primary aim of this study was to evaluate a single mycobacterial antigen, ESAT-6 in the IFN-gamma test for use in skin test-positive cattle. These skin test-positive cattle comprised 51 Mycobacterium bovis-infected animals from tuberculosis-infected herds and 85 non-infected animals from tuberculosis-free herds. The test based on ESAT-6 had a higher specificity than the test based on purified protein derivative (PPD) tuberculin, but this was offset by a small decrease in sensitivity. Use of a lower cut-off in the ESAT-6-based test improved the sensitivity, while still maintaining a very high specificity. A secondary aim in the study was to assess the ESAT-6 and PPD-based tests for detecting bovine tuberculosis in skin test-negative animals from a persistently infected herd. The PPD-based test detected the majority of the lesioned or M. bovis-culture positive animals, while the ESAT-6-based test detected a smaller proportion. The false negatives in the IFN-gamma test from both the skin test-negative and positive groups were predominantly M. bovis-culture positive animals with no visible lesions. The current study has shown that a defined specific antigen such as ESAT-6 can markedly improve the specificity of the IFN-gamma test for re-testing skin test-positive animals. An ESAT-6-based IFN-gamma test could be particularly useful to reduce the false positive rate, yet still maintain an acceptable level of sensitivity.     

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Evaluation of a Whole Blood Glutaraldehyde Coagulation Test for the Detection of Failure of Passive Transfer in Calves

The accuracy of a commercially available whole blood glutaraldehyde clot test in the detection of failure of passive transfer (serum immunoglobulin [Ig]G₁ < 1,000 mg/dL) in neonatal calves was evaluated. Serum samples were obtained from 242 calves ranging in age from 1 to 8 days, and comparisons were made with serum lgG₁ concentrations determined by radial immunodiffusion. Both the sensitivity and specificity of the currently marketed whole blood glutaraldehyde clot test are inadequate for routine diagnostic use. Concerns regarding test sensitivity are the most problematic. Sensitivity varied from 0.41 to 0.00, depending on the choice of test endpoint. Specificity varied from 0.85 to 1.00, depending on the choice of test endpoint. Regression analysis demonstrated that the relationship between serum lgG₁ concentration and the glutaraldehyde clot results, although significant (P< .10), was of negligible biological relevance ( r ² = .034). J Vet Intern Med 1996; 10:82–84. Copyright © 1996 by the American College of Veterinary Internal Medicine .