Longitudinal analysis of acute-phase proteins in saliva in pig farms with different health status

This study assesses the utility of saliva samples to monitor the time course of the acute-phase response to different viruses in pigs under field conditions by using time-resolved immunofluorometric assays (TR-IFMA). A total of 30 pigs from three different farms, located in Southeast Spain, were used. Farm 1 had outbreaks of porcine circovirus type 2, farm 2 had infections with porcine reproductive and respiratory syndrome virus and farm 3 had concomitant infections with both viruses. Serology was used to determine the time of seroconversion of pigs to two different pathogens. The levels of two acute-phase proteins (APPs), C-reactive protein (CRP) and haptoglobin (Hp), were measured in saliva and serum samples and compared with pig's serology. Kinetic curves of both APPs across the study obtained in saliva samples were similar to those of serum, with R of 0.68 and 0.78 for CRP and Hp, respectively. The median CRP and Hp concentrations in saliva were higher around the theorized time of infection, according to previous experimental studies, and at seroconversion of animals. CRP increments were apparent 1 week before the increments obtained in Hp. These findings indicate that salivary APP concentrations, by using TR-IFMA, can be used in longitudinal studies as non-invasive early indicators of health status.     

Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA_359−379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10⁻³ μg/ml and a limit of quantification of 24.5 × 10⁻³ μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.     

An ultrasensitive time-resolved immunofluorometric assay of human epidermal growth factor

We have developed a sandwich-type time-resolved immunofluorometric assay (TR-IFMA) for human epidermal growth factor (hEGF) in body fluids. A two-step solid-phase technique was used. The assay utilizes a polyclonal anti-hEGF attached to the solid phase, and a monoclonal anti-hEGF labeled with Europium (III) as a tracer. The sensitivity of the assay (2.5 pg/ml) is at least 20 times better than what has been achieved by radioimmunoassay (RIA), and the measuring range is much wider: 2.5-5000 pg/ml. The feasibility of TR-IFMA was tested by assaying urine containing large amounts and amniotic fluid containing small amounts (mostly undetectable by RIA) of immunoreactive hEGF. The correlation between urine hEGF concentrations (1-100 ng/ml) measured by RIA and TR-IFMA was good: r = 0.96.     

Cytokine secretion depends on Galalpha(1,3)Gal expression in a pig-to-human whole blood model

Transplants from alpha1,3-galactosyltransferase (Gal) gene-knockout pigs to nonhuman primates are largely protected from hyperacute but not acute humoral xenograft rejection. The present study investigates the role of Gal in cytokine responses using a novel pig-to-human whole blood in vitro model, developed for species-specific analysis of porcine and human cytokines. Porcine (n = 7) and human (n = 27) cytokines were measured using ELISA or multiplex technology, respectively. Porcine aortic endothelial cells from control (Gal(+/+)) and Gal-deficient (Gal(-/-)) pigs were incubated with human lepirudin anticoagulated whole blood from healthy donors. E-selectin expression was measured by flow cytometry. The C3 inhibitor compstatin and a C5aR antagonist were used to study the role of complement. Cytokine species specificity was documented, enabling detection of 2 of 7 porcine cytokines and 13 of 27 human cytokines in one single sample. Gal(+/+) porcine aortic endothelial cells incubated with human whole blood showed a marked complement C5b-9 dependent up-regulation of E-selectin and secretion of porcine IL-6 and IL-8. In contrast, Gal(-/-) cells responded with E-selectin and cytokine expression which was so weak that the role of complement could not be determined. Human IL-6, IL-8, IFN-gamma, MIP-1alpha, MIP-1beta, eotaxin, and RANTES were detected in the Gal(+/+) system, but virtually no responses were seen in the Gal(-/-) system (p = 0.03). The increase in human cytokine release was largely complement dependent and, in contrast to the porcine response, mediated through C5a. Species-specific analysis of cytokine release revealed a marked, complement-dependent response when Gal(+/+) pig cells were incubated with human whole blood, compared with Gal(-/-) cells which induced virtually no cytokine release.     

Solid phase immunoradiometric assay for porcine serum ferritin

1. A solid phase immunoradiometric assay using anti-serum coated polystyrene tubes, is described for the assay of porcine serum ferritin. 2. The mean concentration of ferritin in the serum of both male and female pigs (Sus scrofa) was 12.1 micrograms/l +/- 8.7 micrograms (range less than 1-35 micrograms/l) and no sex differences were observed in 40 pigs from 1 day to 4 years old. 3. Serum ferritin increased with increasing body iron stores in iron loaded pigs as assessed by hepatic iron concentration. 4. The assay is sensitive (detecting less than 1 microgram/l), reproducible, specific and it does not cross-react with human or rat ferritin.     

A multi-herd study shows that saliva is more than a reflection of serum biomarkers in pigs

This study evaluates if biomarkers of porcine health status in saliva samples is a mere reflection of serum to detect disease in pigs under field conditions. Four farms from the same commercial company were included to obtain samples from animals with different pathological conditions. A total of 10 healthy animals and 10–15 animals from each farm with clinical symptoms of the disease were sampled for paired saliva and blood during a veterinary clinical visit. The biomarker panel included acute-phase proteins (APPs), C-reactive protein (CRP), haptoglobin (Hp), an inflammatory marker, adenosine deaminase (ADA), the total antioxidant capacity (TAC), the levels of essential trace elements, copper (Cu) and zinc (Zn), and the measurement of the total protein content (TP). After detailed statistical analysis, the results showed that saliva could replace serum for APP measurements since a good agreement has been observed between the concentrations of APPs in both body fluids. For any other biomarker, no agreement between the concentrations quantified in serum and saliva samples was observed visually. However, salivary ADA and TP concentrations were statistically significantly higher in the diseased, whereas the statistical tests with serum concentrations were inconclusive. Furthermore, greater differentiation between healthy and diseased animals could be observed when the distribution of biomarkers was analysed in saliva than in other serum samples. The diagnostic power to discriminate between healthy and diseased pigs is similar in saliva and in serum samples. Preliminary regression models may offer an optimal combination of biomarkers for disease detection in saliva (Hp, CRP, and TAC) and serum (Hp, CRP, and Cu), which demands less labour, sample, and financial cost for saliva determinations. The contradictory results observed for TAC, Cu, and Zn levels between body fluids indicate a need for further studies. To sum up, saliva-based biomarkers instead of serum-based biomarkers could contribute to more efficient detection of diseased animals.     

An optimised electrochemical biosensor for the label-free detection of C-reactive protein in blood

C-reactive protein (CRP) is an acute phase protein whose levels are increased in many disorders. There exists, in particular, a great deal of interest in the correlation between blood serum levels and the severity of risk for cardiovascular disease. A sensitive, label-free, non-amplified and reusable electrochemical impedimetric biosensor for the detection of CRP in blood serum was developed herein based on controlled and coverage optimised antibody immobilization on standard polycrystalline gold electrodes. Charge transfer resistance changes were highly target specific, linear with logCRPconcentration across a 0.5-50nM range and associated with a limit of detection of 176pM. Significantly, the detection limits are better than those of current CRP clinical methods and the assays are potentially cheap, relatively automated, reusable, multiplexed and highly portable. The generated interfaces were capable not only of comfortably quantifying CRP across a clinically relevant range of concentrations but also of doing this in whole blood serum with interfaces that were, subsequently, reusable. The importance of optimising receptor layer resistance in maximising assay sensitivity is also detailed.     

Anthropometric correlates of C-reactive protein among indigenous Siberians

C-reactive protein (CRP) is an inflammatory marker, which at low-level elevations is associated with increased cardiovascular risk. Although CRP has been extensively investigated in North American and European settings, few studies have measured CRP among non-Western groups. The present study used dried whole blood spot samples to examine high-sensitivity CRP concentrations among the Yakut (Sakha) of Siberia (85 females, 56 males; 18-58 years old). Our goals were: (1) to compare Yakut CRP concentrations with other populations; (2) to investigate sex differences; and (3) to explore anthropometric correlates of CRP. Results indicate that serum equivalent CRP concentrations are similar to those from industrializing nations, lower than US and European values, and greater than Japanese concentrations. Yakut men and women display similar CRP concentrations; however, CRP was significantly higher among men after adjustment for body fat, age, and smoking. Positive associations were documented between CRP and BMI, body fat, and central adiposity.     

Studies on 5alpha-androst-16-en-3-one binding to porcine serum, plasma and testicular cytosolic fraction and to human serum

The present study evaluated whether a specific androstenone-binding protein is present in porcine and human serum, and in the cytosolic fraction of porcine testis. The binding of [(3)H]-androstenone to serum and testicular cytosol was measured in the absence (total binding) and presence (non-specific binding) of unlabelled androstenone. The optimization of the assay is described. As a part of the assay validation, the binding of [(3)H]-dihydrotestosterone ([(3)H]-DHT) to porcine and human serum was also examined. As expected, specific binding of [(3)H]-DHT was detected in human serum, but not in porcine serum. No specific androstenone-binding protein was detected, either in porcine or human serum, or in the cytosolic fraction of porcine testis. The amount of non-specific binding of [(3)H]-androstenone was slightly lower in porcine serum compared to human serum. Between-animal variations in [(3)H]-androstenone binding were studied in plasma samples from 15 animals with androstenone concentrations ranging from 1.1 to 23.1 ng/mL. Mean values+/-standard deviations of binding in these samples were 15.2+/-0.9% for total binding and 15.9+/-0.8% for non-specific bindings. Low between-animal variations indicate that androstenone binding does not affect androstenone accumulation in fat.     

C-reactive protein correlates with macrophage accumulation in coronary arteries of hypercholesterolemic pigs.

A growing body of evidence supports the hypothesis that C-reactive protein (CRP) is a marker of inflammation in coronary artery disease. The purpose of the present study was to test the hypothesis that CRP correlates with macrophage accumulation during the initial stages of coronary vascular disease. Adult male pigs were fed a normal chow (NF) or a high-fat high-cholesterol (HF) diet for 20 wk. After 20 wk, blood was collected for analyses of interleukin-6 (IL-6), CRP, and lipids. After blood collection, the pigs were euthanized and the right coronary arteries (RCA) were harvested and fixed in neutral buffered formalin. Paraffin-embedded sections of RCA were stained immunohistochemically for CRP, scavenger receptor A (SRA), and monocyte chemoattractant protein-1 (MCP-1). All cholesterol fractions were elevated in the HF vs. the NF group (P < 0.05). There was little or no positive staining for CRP, SRA, or MCP-1 in the RCA of NF pigs, but there was extensive staining in lipidladen macrophage foam cells in the HF pigs. Double staining revealed colocalization of CRP with SRA and CRP with MCP-1 in foam cells. Serum IL-6 was below the assay detection limit in all pigs. Serum CRP correlated directly with plasma total cholesterol (R = 0.727, P = 0.041) and accumulation of SRA-positive macrophages (R = 0.938, P < 0.001) in RCA of HF pigs. We conclude that serum CRP correlates with macrophage accumulation and coronary artery disease in hypercholesterolemic pigs.     

The effects of storage time and temperature and anticoagulant on laboratory measurements of canine blood progesterone concentrations

The effects of anticoagulant, storage time, storage temperature, and assay method, on laboratory measurements of blood progesterone concentrations of dogs is unclear; these factors have had a dramatic effect on blood progesterone concentrations in other species (particularly cows). In six experiments, we determined the effects of assay technique (chemiluminescence versus radioimmunoassay (RIA)), storage time, and temperature, as well as the use of heparinized plasma versus serum (coagulated blood) on measured progesterone concentrations of bitches. The studies showed that: (a) RIA measured significantly higher serum progesterone concentration (SPC) than chemiluminescence; (b) refrigeration of whole blood during the first 2 h after sample collection significantly decreased measured SPC; (c) progesterone concentration in heparinized plasma was not affected by storage temperature of whole blood for at least 5 h; (d) refrigeration of whole, clotted blood did not affect SPC, provided that samples were held at room temperature for the first 2 h after collection. These findings are of particular importance when blood samples are collected for determination of the initial rise in SPC that is associated with the LH surge in estrous bitches.     

Effect of C-reactive protein on peritoneal macrophages. II. Human C-reactive protein activates peritoneal macrophages of guinea pigs to release superoxide anion in vitro

The effect of human C-reactive protein (CRP) on macrophage function was studied in an assay of superoxide anion (O2-) production. Peritoneal exudate macrophages (PEM) of guinea pigs exposed in vitro to various doses of CRP for 72 hr resulted in the development of O2- production dose-dependently, measured by increases in superoxide dismutase-inhibitable nitro blue tetrazolium reduction. The O2--producing activity of PEM cultured without CRP, used as a control, decreased markedly in proportion to incubation time. The O2- production by PEM exposed to CRP for 18 hr when control PEM were still high in O2- production, was decreased by larger doses of CRP, while PEM cultured without CRP for 72 hr, when O2- production by control PEM was very low, followed by incubation with CRP for another 18 hr, produced O2- CRP-dose-dependently as in the case of that observed after 72-hr incubation with CRP. These results indicate that CRP is capable of activating macrophages and acts on macrophage function as a modulator. CRP possesses migration inhibitory factor (MIF)-like activity (as reported in the preceding paper) and also macrophage-activating factor (MAF)-like activity, indicating that CRP may play a functional role at the site of inflammation and tissue damage by accumulating and activating macrophages.     

Radioimmunoassay of progesterone in peripheral and placental blood of pregnant rabbits and guinea pigs

Radioimmunoassay of progesterone in systemic and placental blood of pregnant rabbits and guinea pigs. 1. The level of progesterone in pregnant rabbits and guinea pigs serum was measured directly (without extraction) using a RadioImmunoAssay (RIA). 2. Hormonal concentrations in systemic blood were shown to increase with gestational age, being at their highest half-way through pregnancy (16.03 +/- 2.63 ng/ml for rabbits; 319.01 +/- 42.10 ng/ml for guinea pigs) and decreasing at the end of the pregnancy. 3. Progesterone was not detectable in rabbit placental blood, whereas a high level of this hormone was found in guinea pig placental blood, which increased with gestational age. From the 28th to the 56th post-coital day, the level increased from 143.22 +/- 13.15 to 283.30 +/- 36.84 ng/ml. 4. The method used enables to measure correctly progesterone concentrations in rabbit and guinea pig serum without extraction.     

Analysis of serum and whole blood values in relation to helminth and ectoparasite infections of feral pigs in Texas

In the summers of 1996 and 1997, 60 wild pigs (Sus scrofa) were necropsied from three sites in south Texas (USA) to test the hypothesis that serum and whole blood parameters vary significantly (P < or = 0.05) with the prevalence and intensity of parasites infecting wild pigs. We found ten parasite species: five nematodes (Metastrongylus salmi, Metastrongylus pudentotectus, Stephanurus dentatus, Oesophagostomum dentatum, and Physocephalus sexalatus); four ixodid ticks (Amblyomma cajennense, Amblyomma maculatum, Amblyomma americanum, and Dermacentor variabilis); and one trematode (Fascioloides magna). Among juvenile pigs, the intensity of the four species of ticks, collectively, was negatively correlated (P < or = 0.05) with whole blood principal component number one (PC-1); this factor was positively associated with lymphocytes and eosinophils. Lungworm intensity (Metastrongylus spp.) among adult pigs was negatively correlated (P < or = 0.05) with whole blood PC-2; this factor was negatively associated with segmented neutrophils and monocytes. There were no significant correlations found between parasite prevalences and either serum or whole blood principal component factors. The correlations observed between parasite intensities and serum and whole blood parameters generally were weak. Thus, we found no strong evidence that serum and whole blood parameters provided good predictive information on parasite infections in wild pigs for most practical management decisions.     

Oral immunosuppressive medication for growing pigs in transplantation studies

Immunosuppressive (IS) medication is needed to avoid graft rejection in porcine transplantation models. An ideal IS therapy should have no side-effects, but increased susceptibility to infections, disturbed intestinal microflora and toxic effects on organs and tissues are commonly reported. The aim of the present study was to design an IS protocol with tacrolimus and mycophenolic acid to be used for maintenance therapy in the post-transplant period. An eligible whole blood trough value for tacrolimus was 5-15 μg/L. Conventional specific pathogen-free pigs were fitted with an indwelling catheter under general anaesthesia, and after the acclimatization period three groups were formed: group A (n= 4) received 0.15 mg/kg body weight (BW) twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group B (n= 4) received 0.3 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group C (n= 2) did not receive any medication. Daily clinical examinations and analyses of blood concentrations of tacrolimus and glucose were performed. Total and differential white blood cell counts, enzyme activities, bilirubin and electrolyte concentrations were measured every fourth day. At the end of the experiment, the pigs were killed with an overdose of pentobarbital intravenously and a necropsy was performed immediately. All animals seemed to tolerate the IS treatment well. No alterations in their clinical state of health were observed throughout the study and daily weight gain was similar for the three groups. The necropsy did not reveal any pathological findings related to medication. The study showed that 0.25 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid would be an appropriate maintenance dosage for conventional pigs.     

C-reactive protein can influence the proliferation, apoptosis, and monocyte chemotactic protein-1 production of human umbilical vein endothelial cells

C-reactive protein (CRP) has been shown to be closely associated with coronary heart disease. The serum CRP concentrations of chronic periodontitis (CP) patients were increased due to periodontal inflammation. CRP may be a potential key mediator associating CP with coronary heart disease. This study aimed to investigate the effects of CRP on human endothelial cells in vitro. CRP ranging from 0 to 10 μg/mL was adopted to imitate the chronic inflammatory conditions of periodontitis. The influences of CRP on proliferation, apoptosis, and monocyte chemotactic protein-1 (MCP-1) production of human umbilical vein endothelial cells (HUVECs) were studied through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and enzyme-linked immunosorbent assay analysis, respectively. Compared to the blank control, 2.5 and 5.0 μg/mL CRP significantly suppressed cell proliferation by 6.9% and increased apoptosis by 10.2% and 14.6%, respectively (p<0.05). Concentrations of 7.5 and 10.0 μg/mL CRP also induced 2.3% HUVEC proliferation suppression (p>0.05) and significantly increased apoptosis ratio compared to that of the blank control. CRP could promote MCP-1 production of HUVECs in a concentration-dependent manner. The MCP-1 production of 10.0 μg/mL CRP group was about 15.3% higher than that of the control group. It is concluded that low concentrations of CRP, which appears in CP, inhibits cell proliferation, promotes cell apoptosis, and increases MCP-1 production in endothelium, which may initiate self-repairing function of vascular endothelium following vascular injury process.     

Sample handling and stability of hepatocyte growth factor in blood samples

As regards clinical studies performed on hepatocyte growth factor (HGF) during recent years, we have aimed in the present study to investigate the eventual differences in sample handling of this cytokine that might influence the results of serum concentrations. Venous blood from patients with current infectious diseases and controls was used in different sub-studies. Compared with samples separated within one hour, no significant changes in serum HGF levels were observed when whole blood stayed 4, or 24h at 6 degrees C before or 6h in room temperature after separation but HGF levels were significantly higher (P<0.01) when whole blood was kept at room temperature 4 and 24h before separation. Serum HGF was stable up to 20 freeze-thaw cycles. The serum concentrations of HGF were significantly higher than levels in the plasma (19%; P<0.05). A significant increase in serum HGF levels (12%, P<0.05) was observed after shaking the whole blood sample to a visible haemolysis, although the HGF concentration in blood cells was around half of that in serum. HGF tolerated storage at -70 degrees C for at least 4 months. We conclude that standardized methods in sample handling are important in the study of HGF concentrations in blood samples.     

Seroprevalence and risk factors for Taenia solium cysticercosis in rural pigs of northern Peru

Taenia solium is a cestode parasite that causes cysticercosis in both humans and pigs. A serological survey was undertaken to assess the seroprevalence and risk factors associated with porcine cysticercosis in the rural district of Morropon, Peru. Pigs aged between 2 and 60 months were assessed by the Enzyme-linked Immunoelectrotransfer blot (EITB) assay to determine their serological status against porcine cysticercosis in a cross-sectional study. A total of 1,153 pigs were sampled. Porcine seroprevalence was 45.19% (42.31-48.06). The information about the animals and households was analyzed and risk factors associated with seroprevalence were determined by a multivariate logistic regression analysis. In the porcine population, the risk of being seropositive increased by 7% with every month of age (OR 1.07, 95% CI 1.05-1.09), and by 148% for pigs living in East Morropon (OR 2.48, 95% CI 1.82-3.37). Whereas, the presence of latrines in a household decreased the risk of being seropositive by 49% (OR 0.51; 95% CI 0.39-0.67). Sex and rearing system did not represent either risk or protective factors associated with the seroprevalence of porcine cysticercosis. The findings of this study could be used for further development of control programs that might focus on similar population groups within rural communities of developing countries where cysticercosis is endemic.     

Follicular fluid effects on progesterone secretion are not due to follicle-stimulating hormone or steroids

An antiserum raised against porcine follicle-stimulating hormone (FSH) was unable to eliminate the stimulatory action of fluid from large antral porcine follicles on progesterone secretion by granulosa cells from small antral porcine follicles. The same titers of the antiserum were completely effective at eliminating the effect of 2 micrograms of NIH-FSH-P12, whereas maximal stimulation of progesterone secretion was observed with 0.5 micrograms FSH/ml. The androgen and estrogen concentrations measured in charcoal-treated inhibitory follicular fluid from small porcine antral follicles and from stimulatory follicular fluid from large follicles were added separately and together to culture media supplemented with serum to determine if these low concentrations (5 X 10(-11) to 5 X 10(-10) M) of steroids could mimic the actions of follicular fluid on progesterone secretion. Neither the inhibitory nor the stimulatory actions of the follicular fluids could be mimicked by these low concentrations of steroids. Higher concentration of steroids (10(-8) to 10(-7) M range) did stimulate progesterone secretion as reported by others. Our data indicate that the actions of charcoal-treated follicular fluids on granulosa cell progesterone secretion cannot be explained by difference in FSH or steroid contents between the inhibitory and stimulatory fluids and serum.     

非细菌性肺炎血CRP临床意义

目的：评估血CRP测定在小儿非细菌性肺炎中的临床应用价值。方法：对42例支原体、24例衣原体及33例病毒性肺炎共计99例非细菌性肺炎病人进行血CRP值测定。结果：32％非细菌性肺炎病人血CRP值增高;CRP值在支原体肺炎、衣原体肺炎及病毒性肺炎之间差异无显著意义;皿CRP与中性分类显著相关(r=0．628,P=0．011),而与WBC计数及体温不相关。结论：非细菌性肺炎病人血CRP值也可增高,CRP对非细菌性肺炎病原的鉴别能力差。