Validation of the fCAL turbo immunoturbidimetric assay for measurement of calprotectin in porcine and bovine fecal samples.

The concentration of calprotectin in feces is a well-studied marker of gastrointestinal inflammation in humans. However, little is known about fecal calprotectin in farm animals. In this work, we have validated an immunoturbidimetric method for fecal calprotectin (Bühlmann fCAL® turbo assay, Schönenbuch, Switzerland) in porcine and bovine fecal samples. Linearity was evaluated by serial dilution (R2 > 0.97 was obtained for both species). Accuracy was assessed by a recovery study, with results between 80 and 120% for low, medium, and high samples in both species. Intra- and inter-assay variability was <20%. Limit of detection was 6.4 µg/g in pig and 5.3 µg/g in cow. Limit of quantification was 13.4 µg/g (pig) and 11.1 µg/g (cow). Additionally, clinical validation has been included to evaluate the ability of the assay to detect inflammatory status in the intestine under different management conditions. In experiments with porcine, it was found that piglets treated with ZnO had lower concentrations of fecal calprotectin. In a second experiment in bovine, calves with diarrhea had higher concentration of fecal calprotectin. The Bühlmann fCAL® turbo assay is suitable for measurement of calprotectin in porcine and bovine fecal samples. Moreover, fecal calprotectin could be a good biomarker of intestinal inflammation in both species.

Acute phase proteins in cattle and swine: A review.

The major acute phase proteins (APPs) in cattle are haptoglobin (Hp) and serum amyloid A (SAA), and in swine, are Hp, SAA, C-reactive protein (CRP), and Pig major acute phase protein (Pig-MAP). Many methodologic assays are presently available to measure these parameters, which are still being improved to increase their specificity, sensitivity, user-friendliness, and economic availability. In cattle, the main applications are the diagnosis and monitoring of frequent diseases such as mastitis and metritis in dairy cows and respiratory problems in young calves. In pigs, APPs are useful in the control of bacterial and viral infections, and they may be used at the slaughterhouse to monitor subclinical pathologies and improve food safety. The utility of APP in animal production must not be forgotten; optimization of protocols to improve performance, welfare, and nutrition may benefit from the use of APPs. Other sample types besides serum or plasma have potential uses; APP determination in milk is a powerful tool in the control of mastitis, saliva is a non-invasive sample type, and meat juice is easily obtained at the slaughterhouse. Increasing our knowledge of reference intervals and the influence of variables such as age, breed, sex, and the season is important. Finally, worldwide harmonization and standardization of analytical procedures will help to expand the use of APPs.

Validation of new automated turbidimetric immunoassays for the measurement of haptoglobin and inter-α-trypsin inhibitor heavy chain H4 specific for the bovine species.

BACKGROUND: Good strategical programs are required for the early detection of disease even in the absence of evident clinical signs, which is crucial in satisfying animal welfare. Haptoglobin (Hp) and inter-α-trypsin inhibitor heavy chain H4 (ITIH4) are acute phase proteins and good biomarkers of early inflammation in cattle, with plasma levels that significantly increase after injury or infection. OBJECTIVES: We aimed to develop and validate two new immunoturbidimetric methods for Hp and ITIH4. METHODS: Species-specific antibodies were obtained and used to develop the immunoassays. For the Hp assay, antibodies were fixed to latex microparticles to enhance detection. The immunoassays were set up in an automated analyzer to carry out validation studies. Reference intervals were calculated using Reference Value Advisor. RESULTS: The Hp immunoturbidimetric method had a linear analytical range up to 0.40 mg/mL. The limit of detection (LoD) was 0.005 mg/mL, and the limit of quantification (LoQ) was 0.007 mg/mL. Total imprecision was less than 7%. Comparison with ELISA and single radial immunodiffusion (SRID) showed good correlation, whereas the comparison with the colorimetric method showed constant and proportional differences. The ITIH4 immunoassay showed linearity up to 5 mg/mL, and the LoD was 0.002 mg/mL. Total imprecision was less than 6%. Method comparison showed a good correlation with single radial immunodiffusion, both methods being equivalent. Bilirubin, triglycerides, and hemoglobin presented no interference in any of the assays. Reference intervals were 0.007-0.017 mg/mL for Hp and 0.2-0.7 mg/mL for ITIH4 in dairy cows 10 days before parturition. CONCLUSIONS: Immunoturbidimetric methods developed for Hp and ITIH4 can measure basal and increased levels of these proteins, showing adequate precision, accuracy, and robustness.

I-FABP, Pig-MAP and TNF-α as biomarkers for monitoring gut-wall integrity in front of Salmonella Typhimurium and ETEC K88 infection in a weaned piglet model.

Having sensitive serum biomarkers able to determine the structural changes of the small intestine suffering from bacterial digestive diseases could be a valuable tool particularly in piglets at weaning, when intestinal infections are highly prevalent. We evaluated the usefulness of three inflammatory and gut-wall-integrity biomarkers to assess the degree of intestinal histo-morphological damage in piglets. Piglets were orally challenged with Salmonella Typhimurium or enterotoxigenic Escherichia coli (ETEC) to get a variable range of response according to individual variability. Forty-eight piglets were challenged with Salmonella Typhimurium and seventy-two with enterotoxigenic Escherichia coli K88. Clinical signs and faecal score were recorded. At Days 4 and 8 post-inoculation, blood was sampled, animals euthanised and distal ileum dissected. Morphological measures were obtained from the gut tissue, and serum tumour necrosis factor-alpha (TNF-α), pig major acute-phase protein (Pig-MAP) and intestinal fatty acid-binding protein (I-FABP) were determined. Animals developed mild-to-severe diarrhoea after the challenge. When analysing the complete set of analytical results, a high correlation was found among the three serum biomarkers. The most representative morphological indicator was the villus:crypt ratio (V:C), which showed a strong negative correlation with all three biomarkers. Regression analyses between faecal score and the previous variable showed linear relations. When the range of V:C was analysed, based on the quartile distribution of each serum variable, a marked increase in their concentration was observed with greater villus damage. Summarising, the combination of I-FABP, Pig-MAP and TNF-α may be useful for determining the intestinal injury degree and barrier integrity in recently weaned pigs.

Age-Related Serum Biochemical Reference Intervals Established for Unweaned Calves and Piglets in the Post-weaning Period.

The purpose of the present study is to establish the influence of age on serum biochemistry reference intervals (RIs) for unweaned calves and recently-weaned piglets using large number of animals sampled at different ages from populations under different season trials. Specifically, milk replacer (MR)-fed calves from April-July 2017 (n = 60); from December 2016-March 2017 (n = 76) and from April-August 2018 (n = 57) and one group of healthy weaned piglets (n = 72) were subjected to the study. Serum enzymes and metabolites of calves at age of 24 h (24 h after colostrum intake), 2, 5, and 7 weeks from merged trials and piglets at 0, 7, and 14 days post-weaning (at 21, 28, and 35 days of age) were studied. The main variable is age whereas no major trial- or sex-biased differences were noticed. In calves, ALT, AST, GGT, GPx, SOD, NEFAs, triglycerides, glucose, creatinine, total protein, and urea were greatly elevated (p < 0.001) at 24 h compared with other ages; glucose, creatinine, total protein, and urea constantly decreased through the age; cholesterol's lowest level (p < 0.001) was found in 24 h compared with other ages and the levels of haptoglobin remained unchanged (p > 0.1) during the study. In comparison with the adult RIs, creatinine from 24 h, NEFAs from 2 w, GGT from 5 w, and urea from 7 w are fully comparable with RIs or lie within RIs determined for adult. In piglets, no changes were noticed on glucose (p > 0.1) and haptoglobin (p > 0.1) and there were no major changes on hepatic enzymes (ALT, AST, and GGT), total protein, creatinine and urea even though several statistical differences were noticed on 7 days post-weaning. Cholesterol, triglycerides, NEFAs, cortisol and PigMAP were found increased (p < 0.05) while TNF-alpha was found less concentrated (p < 0.001) at 0 days post-weaning compared with other times. Moreover, the RIs of creatinine and GGT are fully comparable with RIs or lie within RIs determined for adult. In conclusion, clinical biochemistry analytes RIs were established for unweaned calves and recently-weaned piglets and among them some can vary at different ages.

Skeletal muscle metabolomics and blood biochemistry analysis reveal metabolic changes associated with dietary amino acid supplementation in dairy calves.

The effects of different amino acid (AA) supplementations of milk protein-based milk replacers in pre-ruminant calves from 3 days to 7 weeks of age were studied. Animals were divided into 4 groups: Ctrl) Control group fed with milk protein-based milk replacer without supplementation; GP) supplementation with 0.1% glycine and 0.3% proline; FY) supplementation with 0.2% phenylalanine and 0.2% tyrosine; MKT) supplementation with 0.62% lysine, 0.22% methionine and 0.61% threonine. For statistical analysis, t-test was used to compare AA-supplemented animals to the Ctrl group. At week 7, body weight and average daily gain (ADG) were measured and blood samples and skeletal muscle biopsies were taken. Blood biochemistry analytes related to energy metabolism were determined and it was shown that MKT group had higher serum creatinine and higher plasma concentration of three supplemented AAs as well as arginine compared with the Ctrl group. GP group had similar glycine/proline plasma concentration compared with the other groups while in FY group only plasma phenylalanine concentration was higher compared with Control. Although the AA supplementations in the GP and FY groups did not affect average daily gain and metabolic health profile from serum, the metabolome analysis from skeletal muscle biopsy revealed several differences between the GP-FY groups and the Ctrl-MKT groups, suggesting a metabolic adaptation especially in GP and FY groups.

Preparation of canine C-reactive protein serum reference material: A feasibility study.

BACKGROUND: The availability of a species-specific reference material is essential for the harmonization of results obtained in different laboratories by different methods. OBJECTIVES: We describe the preparation of a canine C-reactive protein (cCRP) serum reference material containing purified cCRP stabilized in a serum matrix. The material can be used by manufacturers to assign values to their calibrator and control materials. METHODS: The serum matrix was obtained using blood collected from healthy dogs, stabilized and submitted for a delipidation process. The reference material was prepared by diluting purified cCRP in the serum matrix containing 1.0 mol/L HEPES buffer, 3.0 mmol/L calcium chloride, 80,000 kUI/L aprotinin, and 1.0 mmol/L benzamidine hydrochloride monohydrate at a pH of 7.2, and dispensing (0.5 mL) the matrix into vials that were then frozen. RESULTS: The pilot batch of 200 vials was shown to be homogeneous and stable after a stability study at various temperatures and over a total time of 110 days. The prepared material was submitted to an assignment value study. Eight laboratories from different European countries participated by using the same reagents for an immunoturbidimetric method adapted for different analyzers. The obtained cCRP concentration in the reference material was 78.5 mg/L with an expanded uncertainty (k = 2) of 4.2 mg/L. CONCLUSIONS: Canine C-reactive protein serum reference material has been produced that allows harmonization of results obtained by different methods and different laboratories, thus reducing the possibility of errors and misunderstandings.

A new automated turbidimetric immunoassay for the measurement of canine C-reactive protein.

BACKGROUND: In dogs, as in humans, C-reactive protein (CRP) is a major acute phase protein that is rapidly and prominently increased after exposure to inflammatory stimuli. CRP measurements are used in the diagnosis and monitoring of infectious and inflammatory diseases. OBJECTIVES: The study aim was to develop and validate a turbidimetric immunoassay for the quantification of canine CRP (cCRP), using canine-specific reagents and standards. METHODS: A particle-enhanced turbidimetric immunoassay was developed. The assay was set up in a fully automated analyzer, and studies of imprecision, limits of linearity, limits of detection, prozone effects, and interferences were carried out. The new method was compared with 2 other commercially available automated immunoassays for cCRP: one turbidimetric immunoassay (Gentian CRP) and one point-of-care assay based on magnetic permeability (Life Assays CRP). RESULTS: The within-run and between-day imprecision were <1.7% and 4.2%, respectively. The assay quantified CRP proportionally in an analytic range up to 150 mg/L, with a prozone effect appearing at cCRP concentrations >320 mg/L. No interference from hemoglobin (20 g/L), triglycerides (10 g/L), or bilirubin (150 mg/L) was detected. Good agreement was observed between the results obtained with the new method and the Gentian cCRP turbidimetric immunoassay. CONCLUSIONS: The new turbidimetric immunoassay (Turbovet canine CRP, Acuvet Biotech) is a rapid, robust, precise, and accurate method for the quantification of cCRP. The method can be easily set up in automated analyzers, providing a suitable tool for routine clinical use.

Biochemical and proteomic analyses of the physiological response induced by individual housing in gilts provide new potential stress markers.

BACKGROUND: The objective assessment of animal stress and welfare requires proper laboratory biomarkers. In this work, we have analyzed the changes in serum composition in gilts after switching their housing, from pen to individual stalls, which is generally accepted to cause animal discomfort. RESULTS: Blood and saliva samples were collected a day before and up to four days after changing the housing system. Biochemical analyses showed adaptive changes in lipid and protein metabolism after the housing switch, whereas cortisol and muscular markers showed a large variability between animals. 2D-DIGE and iTRAQ proteomic approaches revealed variations in serum protein composition after changing housing and diet of gilts. Both techniques showed alterations in two main homeostatic mechanisms: the innate immune and redox systems. The acute phase proteins haptoglobin, apolipoprotein A-I and α1-antichymotrypsin 3, and the antioxidant enzyme peroxiredoxin 2 were found differentially expressed by 2D-DIGE. Other proteins related to the innate immune system, including lactotransferrin, protegrin 3 and galectin 1 were also identified by iTRAQ, as well as oxidative stress enzymes such as peroxiredoxin 2 and glutathione peroxidase 3. Proteomics also revealed the decrease of apolipoproteins, and the presence of intracellular proteins in serum, which may indicate physical injury to tissues. CONCLUSIONS: Housing of gilts in individual stalls and diet change increase lipid and protein catabolism, oxidative stress, activate the innate immune system and cause a certain degree of tissue damage. We propose that valuable assays for stress assessment in gilts may be based on a score composed by a combination of salivary cortisol, lipid metabolites, innate immunity and oxidative stress markers and intracellular proteins.

Establishment of the biochemical and endocrine blood profiles in the Majorera and Palmera dairy goat breeds: the effect of feed restriction.

Feed restriction, and seasonal weight loss (SWL), are major setbacks for animal production in the tropics and the Mediterranean. They may be solved through the use of autochthonous breeds particularly well adapted to SWL. It is therefore of major importance to determine markers of tolerance to feed restriction of putative use in animal selection. Two indigenous breeds from the Canary Islands, Palmera and Majorera, are commonly used by dairy goat farmers and, interestingly, have different phenotype characteristics albeit with a common ancestry. Indeed, Majorera is well adapted to feed restriction whereas the Palmera is susceptible to feed restriction. In addition, regardless of their importance in dairy production, there are only a limited number of reports relating to these breeds and, to the best of our knowledge, there is no description of their blood metabolite standard values under control conditions or as affected by feed restriction. In this study we analysed the blood metabolite profiles in Majorera and Palmera goats aiming to establish the differential responses to feed restriction between the two breeds and to characterise their metabolite standard values under control conditions. We observed significant differences in creatinine, urea, non-esterified fatty acids (NEFAs), cholesterol, IGF-1 and T3 due to underfeeding. Furthermore, a PCA analysis, revealed that animals submitted to undernutrition could be distinguished from the control groups, with the formation of three separate clusters (Palmera individuals after 22 d of subnutrition (PE22); Majorera individuals after 22 d of subnutrition (ME22) and animals assigned to control conditions (MC0, MC22, PC0 and PC22)), highlighting different responses of the two breeds to undernutrition.

Plasma iron, C-reactive protein, albumin, and plasma fibrinogen concentrations in dogs with systemic inflammatory response syndrome.

OBJECTIVE: To investigate the diagnostic and prognostic value over time of plasma iron compared with the inflammatory markers albumin, C-reactive protein (CRP), and fibrinogen in dogs with systemic inflammatory response syndrome (SIRS). DESIGN: Prospective observational study of sequentially enrolled dogs. SETTING: ICU of a veterinary teaching hospital. ANIMALS: One hundred and sixteen client-owned dogs: 54 dogs with SIRS or sepsis, 42 with focal inflammation, and 20 clinically healthy dogs. MEASUREMENTS AND MAIN RESULTS: Blood samples were obtained on admission in all study groups, and then on alternate days until discharge or death in both inflammation groups. On admission, dogs with SIRS had significantly lower plasma iron (65 ± 5.8 µg/dL, P = 0.001) concentrations than dogs with focal inflammation (89.5 ± 6.2 µg/dL, P = 0.001). Plasma iron, albumin, and CRP effectively discriminated the SIRS/sepsis group from those presenting with focal inflammation with areas under the curve for the receiver operating curves of 0.679, 0.834, and 0.704, respectively. The admission values for these variables did not discriminate survivors from nonsurvivors within the SIRS/sepsis group. However, the magnitude of increase in iron concentration and the decrease in CRP concentration from admission to hospital discharge was higher in survivors than in nonsurvivors within the SIRS/septic group (22.8 vs. 2.51 µg/dL, respectively, P = 0.021 for iron; -67.1 vs. -4.1 mg/L, respectively, P = 0.002 for CRP), resulting in iron and CRP concentrations at hospital discharge for survivors similar to those in the focal inflammation group. CONCLUSION: Hypoferremia is a sensitive marker of systemic inflammation in dogs. In this study, the increase in iron concentrations during the hospitalization period of SIRS/septic dogs was associated with a better prognosis, suggesting that plasma iron in combination with CRP and albumin concentrations might be used to monitor dogs with inflammatory disease processes.

Serum haptoglobin dynamics in pigs vaccinated or not vaccinated against porcine circovirus type 2.

The present work describes the serum haptoglobin (Hp) dynamics in piglets vaccinated and non-vaccinated with a commercial porcine circovirus type 2 (PCV2) vaccine at 3 weeks of age, and its relationship with the average daily weight gain (ADWG). The field study was carried out on two farms (A and B) with a previous clinical history of PCV2-systemic disease (PCV2-SD). The aim of the study was to assess whether Hp could be used as a surrogate marker of PCV2 vaccine efficacy. PCV2 infection was confirmed by quantitative real time PCR (qPCR) in pigs from both farms, but PCV2-SD was only diagnosed in farm A. No statistically significant relation was found between serum Hp concentration and the percentage of qPCR positive animals and the treatment applied (PCV2 vaccination) in both farms. On the other hand, using linear regression analysis, a significant negative correlation between the area under the curve of Hp (AUCHp) and ADWG was observed for farm A (p < 0.00001) and B (p = 0.01). Based on the obtained determination coefficient (R2) values, AUCHp explained 20.0 and 11.6% of the observed ADWG for farms A and B, respectively. The present study supports that the measurement of acute phase proteins may be an indicator of ADWG in pig farms, but it was not apparently feasible to use the serum Hp concentration as a surrogate marker of PCV2 vaccine efficacy.

Proteomic analysis reveals oxidative stress response as the main adaptative physiological mechanism in cows under different production systems.

Three groups of cows representing three ranges of welfare in the production system were included in the study: two groups of Bruna dels Pirineus beef cattle maintained under different management systems (good and semiferal conditions) and a group of Alberes cows, a breed that lives in the mountains (hardest conditions). In order to identify new stress/welfare biomarkers, serum from Bruna cows living in both environments was subjected to DIGE labelling, two-dimensional electrophoresis and MALDI-MS or ion trap MS. Identification was achieved for 15 proteins, which mainly belonged to three biological functions, the oxidative stress pathway (glutathione peroxidase (GPx) and paraoxonase (PON-1)), the acute phase protein family (Heremans Schmid glycoprotein alpha2 (α2-HSG)) and the complement system. Biological validation included the Alberes breed. GPx and PON-1 were validated by an enzymatic assay and found to be higher and lower, respectively, in cows living in hard conditions. α2-HSG was validated by ELISA and found to be reduced in hard conditions. Other biomarkers of the redox status were also altered by living conditions: protein carbonyl content, superoxide dismutase (SOD) and glutathione reductase (GR). Our results show that changes in the redox system are the main adaptation of cows living in challenging environmental conditions.

Association between plasma metabolites and gene expression profiles in five porcine endocrine tissues.

BACKGROUND: Endocrine tissues play a fundamental role in maintaining homeostasis of plasma metabolites such as non-esterified fatty acids and glucose, the levels of which reflect the energy balance or the health status of animals. However, the relationship between the transcriptome of endocrine tissues and plasma metabolites has been poorly studied. METHODS: We determined the blood levels of 12 plasma metabolites in 27 pigs belonging to five breeds, each breed consisting of both females and males. The transcriptome of five endocrine tissues i.e. hypothalamus, adenohypophysis, thyroid gland, gonads and backfat tissues from 16 out of the 27 pigs was also determined. Sex and breed effects on the 12 plasma metabolites were investigated and associations between genes expressed in the five endocrine tissues and the 12 plasma metabolites measured were analyzed. A probeset was defined as a quantitative trait transcript (QTT) when its association with a particular metabolic trait achieved a nominal P value < 0.01. RESULTS: A larger than expected number of QTT was found for non-esterified fatty acids and alanine aminotransferase in at least two tissues. The associations were highly tissue-specific. The QTT within the tissues were divided into co-expression network modules enriched for genes in Kyoto Encyclopedia of Genes and Genomes or gene ontology categories that are related to the physiological functions of the corresponding tissues. We also explored a multi-tissue co-expression network using QTT for non-esterified fatty acids from the five tissues and found that a module, enriched in hypothalamus QTT, was positioned at the centre of the entire multi-tissue network. CONCLUSIONS: These results emphasize the relationships between endocrine tissues and plasma metabolites in terms of gene expression. Highly tissue-specific association patterns suggest that candidate genes or gene pathways should be investigated in the context of specific tissues.

Serum acute phase proteins as biomarkers of pleuritis and cranio-ventral pulmonary consolidation in slaughter-aged pigs.

The purpose of this study was to investigate the relationship between the existence of lung lesions in pigs at slaughter and the concentration of the serum acute phase proteins (APP), haptoglobin (Hp), pig-major acute protein (Pig-MAP) and C-reactive protein (CRP). A total of 24 pig farms were selected out of a larger farm database previously screened to study risk factors associated with pleuritis and cranio-ventral pulmonary consolidation (CVPC) lesions at slaughter-aged pigs in Spain. The farms were classified as "pleuritis negative (P-) or positive (P+)" and as "CVPC negative (M-) or positive (M+)" and divided into four groups according to a 2X2 factorial design (P-M-, P-M+, P+M-, P+M+). Also at slaughter, blood from 20 randomly selected pigs from each farm was collected. Obtained serum samples were used to measure acute phase proteins. All APP concentrations were significantly higher for M+ farms than for M- ones. However, only Hp and Pig-MAP showed significantly higher concentrations for P+ farms than for P- ones. Pig-MAP was the most sensitive biomarker since it was able to clearly discriminate between P-/P+ and M-/M+ groups (p<0.001 in both cases). Hp was an excellent marker for pleuritis and good for CVPC lesions. CRP was able to discriminate for CVPC lesions but not for pleuritis. The present results indicate that Pig-MAP and, possibly Hp, may be used as potential markers to characterise and discriminate respiratory lesions in swine herds at slaughter.

Validation of an immunoturbidimetric method for determination of porcine serum C-reactive protein.

Measurement of porcine C-reactive protein (CRP) in serum is an important tool for monitoring health and welfare in pigs. In this study, an immunoturbidimetric method from Olympus System Reagent (OSR 6147) used to measure human CRP in serum that employ a human traceable calibrator has been evaluated in porcine serum samples. Intra- and inter-assay imprecision were lower than that obtained with the porcine-specific commercially available ELISA. The expected difference in serum CRP between healthy and non-healthy pigs was detected. CRP values measured by the immunoturbidimetric method showed a good correlation with those obtained by ELISA, although differences in absolute CRP values were observed. When an in-house porcine standard was used a better agreement was obtained. In conclusion, the immunoturbidimetric method of Olympus can be used with porcine samples. The easier use of this method should facilitate the implementation of CRP serum determination for diagnostic and prognostic purposes in swine medicine. The results emphasize the need to establish species-specific standard and methods to decrease inter-laboratory discrepancies.

Plasma protein electrophoresis of Trachemys scripta and Iguana iguana.

BACKGROUND: Protein electrophoresis is widely applied in veterinary medicine, but is not used often in reptiles, in part because of lack of reference values. OBJECTIVE: The goals of this study were to compare plasma protein profiles obtained by cellulose acetate electrophoresis (CAE) and agarose gel electrophoresis (AGE), measure precision and examine interference by sample hemolysis, and establish preliminary reference intervals for 2 reptile species. METHODS: Heparinized plasma samples from healthy and diseased adult female Iguana iguana (n=40) and Trachemys scripta (n=60) were analyzed by CAE and AGE. Total protein concentration was measured by the biuret method. Electrophoresis results were compared using Bland-Altman plots and Passing-Bablok regression analysis. Precision and the effects of sample hemolysis were determined. Results from clinically healthy animals were used to determine reference intervals. RESULTS: Five protein fractions were identified in both species, with bisalbuminemia observed in 23/40 iguanas. High correlation was observed between the 2 methods for all fractions, with few proportional and systematic errors. Coefficients of variation were lower using AGE vs CAE and for I. iguana vs T. scripta. Two additional bands were observed in hemolyzed samples from T. scripta; 1 additional band was observed for I. iguana. Minimum and maximum values were reported for healthy I. iguana (n=14) and T. scripta (n=22). CONCLUSIONS: Although both methods are acceptable, the performance of AGE was slightly better than that of CAE for analysis of plasma from reptiles. Furthermore, reptile electrophoretic patterns should be interpreted based on the method used, the species analyzed, and the quality of the plasma sample.

Evaluation of serum haptoglobin and C-reactive protein in dogs with mammary tumors.

BACKGROUND: In veterinary medicine, there is increasing interest in measuring acute phase proteins as a tool in the diagnosis and monitoring of neoplastic diseases. Although mammary neoplasms are the most common type of cancer in dogs, acute phase proteins have not been extensively evaluated in dogs with mammary tumors. OBJECTIVES: The aim of this study was to evaluate serum haptoglobin (Hp) and C-reactive protein (CRP) concentrations in the dogs with mammary tumors and assess their potential association with malignancy. METHODS: A retrospective study of dogs with mammary tumors was performed. Serum concentrations of CRP and Hp were determined in healthy control dogs (n=20) and dogs with mammary tumors before surgery (n=41). Mammary tumors were grouped as carcinomas (n=24), fibrosarcoma (n=1), malignant mixed tumors (n=7), benign mixed tumors (n=6), and adenomas (n=3). CRP and Hp concentrations were compared in dogs with different tumor types and were also compared based on tumor size, lymph node infiltration, skin ulceration, fixation to underlying tissue, and time between tumor identification and removal. RESULTS: Hp concentration was significantly (P<.043) higher in dogs with mammary tumors (median 2.03 g/L, range 0.09-2.94 g/L) compared with controls (1.38 g/L, range 0.08-3.00 g/L), but the range of values overlapped considerably. CRP concentration was higher in dogs with carcinomas (4.70 mg/L, range 0.63-128.96 mg/L) vs controls (2.11 mg/L, range 0.25-6.57 mg/L) (P=.0008) and in dogs with ulcerated skin (14.8 mg/L, range 5.7-128.9 mg/L, n=3) compared with those without ulceration (2.4 mg/L, range 0.11-30.3 mg/L, n=38) (P=.048). CONCLUSIONS: Serum Hp and CRP do not appear to have value in diagnosing or predicting malignancy of mammary tumors in dogs. Higher CRP concentrations in dogs with mammary carcinoma suggest a role for inflammation in this tumor type.

Evaluation of serum cortisol, metabolic parameters, acute phase proteins and faecal corticosterone as indicators of stress in cows.

To assess the validity of laboratory parameters in blood and faeces as indicators of stress in cows, concentrations of cortisol, non-esterified fatty acids (NEFAs), 3-hydroxybutyrate, glucose, triglycerides, cholesterol, serum amyloid A (SAA) and haptoglobin in serum, as well as corticosterone in faeces, were determined in two breeds of cattle (Alberes and Bruna dels Pirineus) under different systems of housing and feeding. Serum cortisol concentrations were markedly elevated in the Alberes group, probably because they were less habituated to human handling. Corticosterone concentrations in faeces were significantly increased in the Bruna dels Pirineus cattle on Alberes pastures. Concentrations of NEFAs and cholesterol were significantly elevated in the Alberes cows, indicating an adrenergic stimulus of lipolysis or the existence of nutritional stress. SAA concentrations were significantly higher in groups living in hardy conditions, whereas there were no significant differences in haptoglobin between the three groups.