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.

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.

Detection of the Accessory Mental Foramina on Human Mandibles Using Cone-beam Computed Tomography: A Systematic Review and Meta-analysis.

INTRODUCTION: This study aimed to perform a systematic review and meta-analysis on accessory mental foramen (AMF) research using cone-beam computed tomographic (CBCT) imaging. METHODS: A systematic review was performed in PubMed, Embase, Thomas Reuter's Web of Science, Scopus, and ScienceDirect databases according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis statement. Articles focusing on AMF prevalence and location using CBCT imaging were selected without language restrictions. Studies reporting pooled results only or presenting any pathology in the area surrounding the mental foramen (MF) were excluded. A meta-analysis using random effects was performed. RESULTS: The present meta-analysis included a total of 46 articles involving 21,761 subjects. The overall pooled AMF prevalence was 7.87% (95% confidence interval [CI], 6.69-9.24) in subjects and 4.75% (95% CI, 3.79-5.95) in hemimandibles (n = 31,158). AMF presence was most commonly unilateral, reaching 90.15% (95% CI, 82.98-94.49). AMFs were significantly more frequent in right hemimandibles (χ2 = 5.20, P < .05) and were most commonly located posterior and inferior to the MF. However, AMFs superior to the MF were also observed in 47.43% (95% CI, 38.45-56.58) of cases. The studies conducted over the last 3 years showed significantly higher AMF prevalence levels (χ2 = 5.12, P < .05). CONCLUSIONS: Our meta-analysis demonstrates that AMF prevalence is considerable and should not be underestimated. AMFs are most frequently located in right hemimandibles. The presence of AMFs superior to the MF is frequent. Around 3% of people present superior AMFs. This fact puts those patients at greater risk for injury when performing periapical surgery in this area.

Neurobiology of environmental enrichment in pigs: hanges in monoaminergic neurotransmitters in several brain areas and in the hippocampal proteome.

Environmental enrichment in porcine farms improves animal welfare and leads to better public acceptance. To better understand the neurological mechanisms of the response to environmental enrichment, monoaminergic neurotransmitters were quantified in several brain areas from pigs after eight weeks of housing in barren or enriched conditions. Furthermore, iTRAQ labelling combined with LC-MS/MS was used to identify differentially abundant proteins in the hippocampus. Blood biochemical parameters related with stress and welfare were measured. Pigs under enriched conditions showed a decrease in plasma cortisol and lactate. The decrease in noradrenaline in the prefrontal cortex and amygdala, a general decrease in the dopaminergic system and an increase of serotonin in the striatum indicate a lower response to stress in enriched conditions. In the proteomic analysis, 2304 proteins were identified, of which 56 were differential between housing groups (46 upregulated and 10 downregulated). Bioinformatics analysis revealed that they were mainly related to ribosome, translation, microtubules and metabolic mitochondrial processes, indicating that pigs under enriched environments have higher abundance of proteins related to protein synthesis and neuronal activity. Together with previous behavioural studies, our results suggest that environmental enrichment provides a less stressful environment and that pigs cope better with stress conditions like the slaughterhouse. SIGNIFICANCE: Animal welfare has become an important aspect for the sustainability of animal production. The modification of the environment by enriching it with rooting materials and wider space allowance is known to have a positive effect on pigs' welfare. Searching for the underlying neurobiological mechanisms, we found that housing in an enriched environment increased the abundance of proteins related to protein synthesis, microtubule assembly, vesicle-mediated transport and energy metabolism in the hippocampus of pigs. Likewise, changes in the neurotransmitter profile in several brain areas were compatible with a better response to stress. This study expands the knowledge about the biological basis of animal welfare-promoting actions.

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.

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.

Dissipation of Fungicide Residues during Winemaking and Their Effects on Fermentation and the Volatile Composition of Wines.

The effects of four fungicides commonly used for the control of fungal diseases in vines and grapes in the course of winemaking were tested. The concentration of fungicide residues was monitored throughout the process to establish their kinetics of dissipation. In all cases the percentages of dissipation were >68%, which shows the detoxificant effect of the winemaking process. On the other hand, the effect of the fungicide residues on the aroma composition of Tempranillo red wines was tested. To evaluate possible modifications on the aroma profile of wines, seven odorant series (ripe fruits, fresh fruits, lactic, floral, spicy, vinous, and herbaceous) were built from the odor activity values (OAVs) obtained for each volatile compound. Ripe fruits and fresh fruits were the major aromatic attributes in all Tempranillo red wines. These two odorant series registered the highest variations in their total OAVs with respect to the control wine, especially with the application of boscalid + kresoxim-methyl into vines, leading to a decrease in the ripe fruit and fresh fruit nuances of the resulting wines. Moreover, when the effect of these fungicides on the aroma of Tempranillo red wines was compared throughout two years (2012 and 2013), wines elaborated from grapes treated in the field with boscalid + kresoxim-methyl in 2013 displayed the highest variation in aroma profile with respect to control wine.

Effect on the aroma profile of Graciano and Tempranillo red wines of the application of two antifungal treatments onto vines.

The effect of two antifungals (boscalid+kresoxim-methyl and metrafenone) applied onto vines under Good Agricultural Practices (GAPs) on the volatile composition of Tempranillo and Graciano red wines was studied. Changes in aroma profile in the wines were assessed from the combined odour activity values (OAVs) for the volatile compounds in each of seven different odorant series (viz., ripe fruits, fresh fruits, lactic, floral, vinous, spicy and herbaceous). Graciano wines obtained from grapes treated with the antifungals exhibited markedly increased concentrations of varietal volatile compounds (monoterpenes and C13-norisoprenoids) and aldehydes, and decreased concentrations of acetates and aromatic alcohols. By contrast, the concentrations of volatile compounds in Tempranillo wines showed different changes depending on the fungicide applied. Also, the aroma profiles of wines obtained from treated grapes were modified, particularly the ripe fruit nuances in Graciano wines. The OAV of this odorant series underwent an increase by more than 60% with respect to the control wine as a result of the increase of ß-damascenone concentration (which imparts wine a dry plum note). The aroma profile of Tempranillo red wines containing metrafenone residues exhibited marked changes relative to those from untreated grapes.

Sensory description of sweet wines obtained by the winemaking procedures of raisining, botrytisation and fortification.

The effect of winemaking procedures on the sensory modification of sweet wines was investigated. Garnacha Tintorera-based sweet wines were obtained by two different processes: by using raisins for vinification to obtain a naturally sweet wine and by using freshly harvested grapes with the stoppage of the fermentation by the addition of alcohol. Eight international sweet wines were also subjected to sensory analysis for comparative description purposes. Wines were described with a sensory profile by 12 trained panellists on 70 sensory attributes by employing the frequency of citation method. Analysis of variance of the descriptive data confirmed the existence of subtle sensory differences among Garnacha Tintorera-based sweet wines depending on the procedure used for their production. Cluster analysis emphasised discriminated attributes between the Garnacha Tintorera-based and the commercial groups of sweet wines for both those obtained by raisining and by fortification. Several kinds of discriminant functions were used to separate groups of sweet wines--obtained by botrytisation, raisining and fortification--to show the key descriptors that contribute to their separation and define the sensory perception of each type of wine.

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.

Root resorption in chronic periodontitis: a morphometrical study.

BACKGROUND: It has been previously observed that teeth with severe chronic periodontitis experience more transient external root resorptions than teeth with less severe disease. METHODS: Sixty-six teeth diagnosed with different levels of chronic periodontitis in 48 patients were observed with a light microscope. Main characteristics of teeth with root resorptions, as well as percentages of area and volume resorbed on each radicular third of the teeth, were determined. RESULTS: Over 80% (53) of the teeth exhibited resorption: 46.67% in group 1 (slight periodontitis); 85% in group 2 (moderate periodontitis); and 93.55% in group 3 (severe periodontitis). In group 1, the average resorption length was 295.65 microm and the average area, 11,212.45 microm2. In group 2 these measures were 519.09 and 25,678.102, respectively. In group 3, the average length was 675.95 microm, and the average area 36,925.06 microm2. Except for the resorbed volume on the apical third, the other resorption percentages were higher in group 3 (severe periodontitis) and smaller in the group 1 (slight periodontitis), but the location of the largest resorption percentages varied among the three groups. CONCLUSIONS: The severity of periodontitis plays a major role in the presence and extent of transient external root resorption. In the three groups, most of the teeth showed resorptions on the apical third, but severity of periodontitis determined the location of the largest percentages of area and volume resorbed.