Pru p 3, a marker allergen for lipid transfer protein sensitization also in Central Europe.

In the Mediterranean area, lipid transfer proteins (LTPs) are important causes of plant-food allergies often associated with severe allergic reactions. There, peach LTP (Pru p 3) seems to be the primary sensitizer, whereas in Central Europe, little is known about the importance of LTP sensitization. In this region, allergen extract-based diagnosis is often complicated by co-sensitization to Bet v 1, the major birch pollen allergen, its cross-reactive food allergens, and profilins. We investigated the role of LTP sensitization in Central European patients displaying strong allergic reactions to plant-derived food. Analysis of IgE reactivity revealed that ten of thirteen patients were sensitized to Pru p 3, nine to Bet v 1, and two to profilin. Our results showed that LTP sensitization represents a risk factor for severe allergic symptoms in Central Europe. Furthermore, the strong IgE reactivity detected in immunoblots of plant-food extracts indicated that Pru p 3 can be used as a marker allergen for LTP sensitization also in Central European patients.

The levels of zearalenone and its metabolites in plasma, urine and faeces of horses fed with naturally, Fusarium toxin-contaminated oats.

Concentration profile of zearalenone (ZON) and its metabolites in plasma, urine and faeces samples of horses fed with Fusarium toxin-contaminated oats is described. In plasma, ß-zearalenol (ß-ZOL) was detected at high levels on day 10 of the study (3.21-6.24 µg/l). ß-Zearalenol and α-zearalenol were the major metabolites in urine. Zearalenone, α-ZOL and ß-ZOL were predominantly found in faeces. Zearalanone could also be detected in urine (1.34-5.79 µg/l) and faeces (1 µg/kg). The degree of glucuronidation was established in all sample types, approximately 100% in urine and plasma. Low per cent of glucuronidation (4-15%) was found in faeces samples. The results indicate the main conversion of ZON into ß-ZOL in horse. This finding could explain why horse is not susceptible to ZON in comparison with swine which produce α-ZOL as a predominant metabolite.

Regulation of volatile and non-volatile pheromone attractants depends upon male social status.

We investigated the regulation of chemical signals of house mice living in seminatural social conditions. We found that male mice more than doubled the excretion of major urinary proteins (MUPs) after they acquired a territory and become socially dominant. MUPs bind and stabilize the release of volatile pheromone ligands, and some MUPs exhibit pheromonal properties themselves. We conducted olfactory assays and found that female mice were more attracted to the scent of dominant than subordinate males when they were in estrus. Yet, when male status was controlled, females were not attracted to urine with high MUP concentration, despite being comparable to levels of dominant males. To determine which compounds influence female attraction, we conducted additional analyses and found that dominant males differentially upregulated the excretion of particular MUPs, including the pheromone MUP20 (darcin), and a volatile pheromone that influences female reproductive physiology and behavior. Our findings show that once male house mice become territorial and socially dominant, they upregulate the amount and types of excreted MUPs, which increases the intensities of volatiles and the attractiveness of their urinary scent to sexually receptive females.

Author Correction: Regulation of volatile and non-volatile pheromone attractants depends upon male social status.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Effects of B-trichothecenes on luminal glucose transport across the isolated jejunal epithelium of broiler chickens.

Trichothecenes are closely-related sesquiterpenoids (ring structure) with a 12, 13 epoxy ring and a variable number of hydroxyl, acetyl or other substituents. In chickens, D-glucose and amino acid absorption occurs via carrier-mediated transport. Recently, it has been observed that deoxynivalenol (DON) alters the gut function and impairs glucose and amino acid transport in chickens. The purpose of this work was to determine the effects of different B-trichothecenes [DON, Nivalenol (NIV), 15-Ac-DON and Fusarenon X (FUS X)] on intestinal carrier-mediated sodium co-transport of D-glucose in the small intestine of broiler chickens. Intestinal transport was determined by changes in the short-circuit current (Isc), proportional to ion transmembrane flux, in the middle segment of the jejunum of broilers with the Ussing chamber technique. D-glucose produced an increase of the Isc, and this effect was reverted by different B-trichothecene mycotoxins, indicating that the glucose induced Isc was altered by B-trichothecenes. The addition of glucose after pre-incubation of the tissues with B-trichothecenes had no effect (p > 0.05) on the Isc, suggesting that B-trichothecenes afflicted the Na(+)-D-glucose co-transport. However, FUX had no obvious effect on the measured parameters. It could be concluded from the present study that the glucose co-transporter activity appears to be more sensitive to DON, NIV and 15-Ac-DON suppression than by FUS X in the jejunum of broilers.

In vitro effects of deoxynivalenol on small intestinal D-glucose uptake and absorption of deoxynivalenol across the isolated jejunal epithelium of laying hens.

Deoxynivalenol (DON) is a common mycotoxin contaminant in feedstuffs. It has been shown to cause diverse toxic effects in animals. The aim of the present study was to evaluate the effects of DON on the glucose transport capacity in chickens' jejunum and to investigate the permeation of DON itself by the Ussing chamber technique. Glucose uptake into chicken jejunal epithelia was measured after the addition of 200 mumol/L of (14)C-labeled glucose to the mucosal solution. Glucose uptake under control condition was 3.28 +/- 0.53 nmol/cm(2) x min. The contribution of sodium glucose-linked transporter 1 (SGLT-1) to total glucose uptake was estimated by inhibiting SGLT-1 with phlorizin (100 micromol/L). In the presence of phlorizin, glucose uptake was reduced (P < 0.05) to 1.21 +/- 0.19 nmol/cm(2) x min. Deoxynivalenol decreased (P < 0.05) the glucose uptake in the absence of phlorizin to 1.81 +/- 0.24 nmol/cm(2) x min but had no additional effect on the glucose uptake in the presence of phlorizin (0.97 +/- 0.17 nmol/cm(2) x min). Mucosal-to-serosal permeation of DON was proportional to the initial DON concentration over a concentration range from 1 to 10 mug/mL on the mucosal side. Apparent permeability at 10 microg/mL of DON measured 60 to 90 min after DON application was 1.7 x 10(-05) cm/s. It can be concluded that DON (10 mg/L) decreases glucose uptake almost as efficiently as phlorizin. The similarity between the effects of phlorizin and DON on glucose uptake evidences their common ability to inhibit Na(+)-D-glucose cotransport. In addition to local effects, DON can be absorbed from the jejunum. A predominant part of DON passes across the chicken intestinal epithelium by passive diffusion, which is likely on the paracellular pathway. The results imply that the exposure to DON-contaminated feeds may negatively affect animal health and performance by local (i.e., inhibition of intestinal SGLT-1) and systemic effects.

Detection and first characterization of an uncommon haptoglobin in porcine saliva of pigs with rectal prolapse by using boronic acid sample enrichment.

Salivary glycoprotein profiles, obtained after boronic acid enrichment, were studied for the first time in pigs in order to search for specific overall alterations related to acute inflammatory condition. Five healthy pigs and five pigs suffering from rectal prolapse were used, and the levels of acute phase proteins were measured to determine the degree of inflammation of the animals. The enriched glycoprotein profiles, achieved by two-dimensional gel electrophoresis (2DE) were statistically evaluated and spots that appeared differentially regulated between states were subjected to MS analysis for protein identification. Spots from three unique proteins were identified: carbonic anhydrase VI (CA VI), α-1-antichymotrypsin and haptoglobin (Hp). CA VI appeared as two adjacent horizontal spot trains in the glycoprotein profile of healthy animals in its regular isoelectric points (pI). One spot of α-1-antichymotrypsin was found in saliva from pigs with rectal prolapse in an unusual basic pI, and was considered as a breakdown product. Hp was identified as several spot trains in saliva from pigs with rectal prolapse in an unusual alkaline pI and was consequently further investigated. SDS-PAGE and 2DE of paired serum and saliva samples combined with Western blot analysis showed that the unusual Hp position observed in saliva samples was absent in serum. Furthermore, N-glycans from serum and saliva Hp glycopatterns were evaluated from SDS-PAGE Hp bands and showed that the serum N-glycan distribution in Hp ß-chain was comparable in quantity and quality in both groups of animals. In saliva, no Hp ß-chain derived N-glycans could unambiguously be identified from this sample set, thus needing further detailed investigations in the future.

Determination of zearalenone and its metabolites in urine, plasma and faeces of horses by HPLC-APCI-MS.

The paper describes a method for the sensitive and selective determination of zearalenone and its metabolites in urine, plasma and faeces of horses by high performance liquid chromatography and atmospheric pressure chemical ionisation (APCI) mass spectrometry (MS). While only one step sample clean-up by an immunoaffinity column (IAC) was sufficient for plasma samples, urine and faeces samples had to be prepared by a combination of a solid-phase extraction (SPE) and an immunoaffinity column. The method allows the simultaneous determination of zearalenone and all of its metabolites; alpha-zearalenol, beta-zearalenol, alpha-zearalanol, beta-zearalanol and zearalanone. Dideuterated zearalanone was used as internal standard for quantification and the study of the matrix effect. Recovery rates between 56 and slightly above 100% were achieved in urine samples, and more than 80% in plasma and faeces samples. The limits of detection ranged from 0.1-0.5 microg/l or microg/kg, the limits of quantification from 0.5-1.0 microg/l or microg/kg. The practical use of the method is demonstrated by the analysis of spiked and naturally contaminated urine, plasma and faeces of horses.

Effect of addition of a probiotic microorganism to broiler diets contaminated with deoxynivalenol on performance and histological alterations of intestinal villi of broiler chickens.

An experiment was conducted to study the effects of deoxynivalenol (DON) on the performance of broilers, organ weights, and intestinal histology and to evaluate the efficacy of a probiotic feed additive (PB, Eubacterium sp.) with the ability to deepoxidize DON. Two hundred seventy-seven 1-d-old broiler chicks were randomly assigned to 1 of the 3 dietary treatments for 6 wk. The dietary treatments were 1) control; 2) artificially contaminated diets with 10 mg of DON/kg of diet; 3) DON-contaminated diets plus probiotic feed additive (DON-PB). The BW and the efficiency of feed utilization were not adversely affected (P > 0.05) by the inclusion of DON in the diets. A slight improvement in feed intake and BW gain over the course of the experiment was observed in broilers fed DON-PB with no change in feed efficiency. The absolute or relative organ weights were not altered (P > 0.05) in broilers fed the diet containing DON compared with controls and the DON-PB group. The absolute liver weights were numerically increased (P < 0.1) for broilers receiving the diet containing DON-PB. There were no significant differences in the absolute and relative weights of the gizzard, duodenum, pancreas, heart, and spleen. However, the absolute and relative weights of the jejunum and cecum were increased for DON-PB-fed broilers compared with the controls and DON group. No pathological lesions were found in the gut of birds fed DON-contaminated diets during the feeding trial, but mild intestinal changes were observed. The DON altered small intestinal morphology, especially in the duodenum and jejunum, where villi were shorter and thinner (P < 0.05). The addition of the eubacteria to the DON-contaminated feed of the broilers effectively alleviated the histological alterations caused by DON and led to comparable villus length as in the control group. In conclusion, diets with DON contamination below levels that induce a negative impact on health and performance could affect small intestinal morphology in broilers. The histological alterations caused by DON were reduced by supplementing the DON-containing diets with PB. This indicates that in case of DON contamination of feedstuffs, the addition of PB would be a proper way to counteract the possible effects caused by this mycotoxin.

In vitro effects of deoxynivalenol on electrical properties of intestinal mucosa of laying hens.

Deoxynivalenol (DON) is common in European cereal grains, and of all the trichothecenes, poses the greatest problems to animal health. The present study investigated the effects of DON on electrophysiological parameters in laying hens' jejunum mounted in Ussing chambers. In vitro studies were performed to measure the effects of different luminal concentrations of DON (0.5, 1, 5, and 10 microg/mL) on the transmural potential difference, electrical tissue resistance, and electrogenic ion flux rates (short-circuit current, Isc) across the isolated gut mucosa. Deoxynivalenol did not alter (P > 0.05) the transmural potential difference. Resistance was higher (P < 0.05) in the tissues exposed to DON compared with basal values. Deoxynivalenol caused a dose-dependent decrease in Isc (P < 0.05). To investigate the mechanism of action of DON, amiloride (a specific inhibitor for Na+ transport) was added after incubation of the tissue with DON. Amiloride did not decrease (P > 0.05) Isc under these conditions. This may indicate that DON inhibited the Na+ transport before addition of amiloride, which did not then show further inhibitory effects. The addition of D-glucose (5 mmol/L) on the luminal side of the isolated mucosa increased (P < 0.05) Isc, and this effect was reversed by phlorizin (a specific inhibitor of sodium/glucose transporter 1), indicating that the glucose-induced Isc increase may be due to Na+-D-glucose cotransport. In our study, DON decreased (P < 0.05) the glucose-induced Isc in a similar way to phlorizin. The remarkable similarity between the effects of phlorizin and DON on electrical properties seemed to be consistent with their common ability to inhibit Na+-D-glucose cotransport. In conclusion, DON decreased the Isc via inhibition of Na+ transport. The effect on intestinal electrical properties was similar to that of phlorizin after addition of glucose, suggesting that DON may inhibit Na+-D-glucose cotransport. The inhibition of Na+ transport and Na+-D-glucose cotransport are important mechanisms of DON toxicity in the intestine of laying hens.

Effects of luminal deoxynivalenol and L-proline on electrophysiological parameters in the jejunums of laying hens.

Most amino acids are cotransported with sodium. Deoxynivalenol (DON) decreases glucose absorption in the chicken small intestine in vivo and in vitro, and this effect is apparently mediated by the inhibition of the sodium D-glucose cotransporter. DON could selectively modulate the activities of other intestinal transporters. In order to assess this hypothesis, a study was conducted to characterize the in vitro effects of DON in the presence of mucosal amino acids, using L-proline as a model, on the electrophysiological parameters in the jejunums of laying hens. L-Proline (mucosal concentration of 1 mmol/L) was added to a stripped proximal part of jejunum sheets mounted in Ussing chambers in Ringer buffer, and the electrical properties were measured. The transmural potential difference (PD) was nearly constant between the treatments. The tissue resistance (Rt) was higher (P < 0.05) in the tissues exposed to DON compared with basal values and the values after addition of L-proline. Addition of L-proline on the luminal side of the isolated mucosa increased (P < 0.05) the short circuit-current (Isc), and it decreased (P < 0.05) after addition of DON, indicating that the proline-induced Isc was altered by DON. The addition of proline after incubation of the tissues with DON had no effect (P > 0.05) on PD or Rt. Proline did not increase the Isc under these conditions. DON decreased (P < 0.1) the Isc after addition of proline, indicating that DON inhibited the Na+-amino acid co-transport. We concluded from the present study that the amino acid cotransporter activity appears to be highly sensitive to DON suppression.

Determination of cholesterol oxides in processed food using high-performance liquid chromatography-mass spectrometry with atmospheric pressure chemical ionisation.

The present work describes the development and application of an on-line atmospheric pressure ionisation (APCI) LC-MS interface for the simultaneous determination of seven toxicologically relevant cholesterol oxides (7alpha-hydroxycholesterol, 7beta-hydroxycholesterol, 25-hydroxycholesterol, 7-ketocholesterol, 5,6alpha-, 5,6beta-epoxycholesterol and cholestan-3beta,5alpha,6beta-triol). The HPLC method has been optimised to reach better separation of all tested compounds. The influences of APCI parameters (nebulising temperature, cone voltage, source temperature) on signal intensity and fragmentation pattern were investigated for all tested cholesterol oxides compounds. This is the first report on optimisation and determination of two compounds 7alpha-hydroxycholesterol and 5,6beta-epoxycholesterol in processed food using LC-MS. After extraction with hexane, clean-up was carried out using solid-phase extraction on a silica column. For the chromatographic separation of cholesterol oxides an Aquasil C18 column was used with acetonitrile-methanol (60:40) as mobile phase. For the first time we report the use of such a C18 column with a relatively hydrophilic nature for the separation of cholesterol oxides. APCI-MS detection was then applied in selected ion monitoring and positive ion modes by using the molecular ions and the main fragments. The developed method shows good linearity, high repeatability and good recovery for all tested cholesterol oxides. The method was applied for determination of seven selected cholesterol oxidation products in different foodstuffs such as butter, butteroil, lard and egg powder.

Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation.

A new, rapid and sensitive method has been developed for the determination of nivalenol (NIV) and deoxynivalenol (DON) by using HPLC in combination with an atmospheric pressure chemical ionization (APCI)-interface and a single quadrupole mass spectrometer. Different LC and MS parameters have been optimized prior to this in order to obtain better results and sensitivity. The effect of nebulizing temperature on the sensitivity and fragmentation of NIV and DON in an APCI interface was investigated. Also, the influence of the cone voltage on the fragmentation pattern was studied, which was shown to have a tremendous effect. Furthermore, the effect of modifiers such as ammonium acetate, acetic acid and ammonia on the ionisation yield of the above substances have been investigated. The extraction was carried out using acetonitrile-water. A two step purification was then applied on two different Mycosep clean up columns. We have used a modified, rapid and isocratic HPLC method combined with a negative ion APCI-MS for the separation and quantitative determination of NIV and DON in wheat extract. An RP C18 column was used for the separation of selected compounds in wheat extract with water-acetonitrile-methanol (82:9:9, v/v/v) at a flow-rate of 1 ml/min without a split. Calibration curves show good linearity and reproducibility. The detection limit and precision were determined for NIV and DON. Both compounds could be detected down to microg/kg level in wheat using selected ion monitoring of the [M-H]- ions and the main fragments.

Simultaneous quantification of A-trichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical ionisation mass spectrometry.

An approach for simultaneous determination of the main type A-trichothecenes by liquid chromatography and atmospheric pressure chemical ionization mass spectrometry is described. Parameters for coupling of LC-MS such as cone voltage, nebulizing temperature and the LC flow-rate, were optimized to provide detection of mycotoxins with maximum sensitivity. Furthermore, the effects of cone voltage and temperature on the fragmentation pattern of the tested toxins were studied. Main type A-trichothecenes such as T-2 Toxin, HT-2 Toxin, acetyl T-2 Toxin, diacetoxyscirpenol, monoacetoxyscirpenol (15-acetoxyscirpenol) and neosolaniol were separated on a reversed-phase narrow bore C18 column, using a linear gradient and a flow-rate of 0.3 ml/min. Mass spectra were obtained in positive ion mode for confirmation and quantitation. The method involves extraction and purification of toxins by using multifunctional Mycosep columns. Deuterated T-2 Toxin was used as an internal standard. A linear working range between 80 and 500 microg/kg in matrix with an acceptable correlation coefficient was observed. The developed method was validated by using a blank oats sample. The detection limit in the matrix was found to be between 50 and 85 microg/kg in selected ion mode for all tested A-trichothecenes. Recovery data were found to be between 77 and 101%. Within run and day-to-day precision were determined as having comparable levels to those found using GC methods. Furthermore, the matrix effect was investigated by comparing the internal standard versus the external standard method in quantification studies. In addition, the developed method was applied for the analysis of naturally contaminated oats, maize, barley and wheat samples.

Simultaneous determination of major B-trichothecenes and the de-epoxy-metabolite of deoxynivalenol in pig urine and maize using high-performance liquid chromatography-mass spectrometry.

A selective analytical method based on high-performance liquid chromatography (HPLC), combined with atmospheric pressure chemical ionisation (APCI-) mass spectrometry (MS), has been developed for simultaneous determination of B-trichothecenes and the major metabolites of deoxynivalenol. The method allows simultaneous analysis of nivalenol (NIV), deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), fusarenon X (Fus-X) and de-epoxydeoxynivalenol (DOM-1). The method is based on one-step sample clean-up using a multifunctional MycoSep column. A linear gradient mobile phase system, consisting of water:acetonitrile:methanol (H2O:ACN:MeOH) at a flow-rate of 1 ml/min, and a Polar-RP C18 column, were utilised to obtain the best resolution of all tested compounds along with column and equilibrating within 30 min. Dexamethasone (Dex) was used as internal standard. The developed method shows good repeatability for inter- and intra-day precisions as well as good linearity of calibration curves (r2 ranged from 0.9936 to 0.9998). Average recoveries for tested compounds in both matrices have been determined ranging from 63.7 to 102.3% and limit of quantification (LOQ) ranged from 25 to 150 ng/g. The utility and practical impact of the method is demonstrated using contaminated pig urine and maize samples.

Effects of deoxynivalenol on general performance and electrophysiological properties of intestinal mucosa of broiler chickens.

A feeding trial was conducted to evaluate the effects of diets contaminated with deoxynivalenol (DON on the performance of broilers and on the electro-physiological parameters of the gut. The control group was fed the starter and finisher diets without addition of DON. Another group of broilers was fed the starter and finisher diets with 10 mg/kg DON, whereas another group was fed the DON-contaminated diets supplemented with a microbial feed additive (Eubacterium sp.). The diets were provided ad libitum for 6 wk. DON had no effect (P > 0.05) on feed consumption, feed conversion, or body weight. The effect of DON on the electrophysiological parameters of the jejunum was studied in vitro using isolated gut mucosa in Ussing chambers. At the end of the feeding period, 7 birds from each group were killed, and the basal and glucose stimulated transmural potential difference (PD), short-circuit current (Isc), and electrical resistance (R) were measured in the isolated gut mucosa to characterize the electrical properties of the gut. The transmural PD did not differ (P > 0.05) among groups. The tissue resistance was greater (P < 0.05) in birds receiving DON and the microbial feed additive than in the controls and DON group. Addition of D-glucose on the luminal side of the isolated mucosa increased (P < 0.05) Isc in the control and DON-probiotic (Eubacterium sp.; PB) groups, whereas it decreased (P < 0.05) in the DON group indicating that the glucose-induced Isc was altered by DON. Addition of the eubacteria to the DON contaminated feed of the broilers led to electrophysiological properties in the gut that were comparable with those of the control group. It could be concluded that 10 mg/kg DON in the diet impaired the Na(+)-D-glucose cotransport in the jejunum of broilers. In the absence of clinical signs, and without impaired performance, DON appeared to alter the gut function of broilers. The addition of Eubacterium sp. may be useful in counteracting the toxic effects of DON on intestinal glucose transport.

Feeding laying hens seal blubber oil: effects on egg yolk incorporation, stereospecific distribution of omega-3 fatty acids, and sensory aspects.

Seventy-two 26-wk-old Single Comb White Leghorn laying hens were randomly assigned to 36 cages (2 per cage) in a 3-orthogonal 4 x 4 latin square, with the fourth row suppressed, to assess the effect of feeding refined seal blubber oil (SBO, containing 22.2% omega-3 fatty acids) on the fatty acid composition and position in the egg yolk lipids. The experiment was conducted over a period of 9 wk. Eggs were collected and numbered, and the weights were recorded for each week and cage. Eggs collected at wk 5 and 9 were used for total lipid, lipid class, fatty acid, and positional analyses. Sensory evaluation was carried out on eggs collected at wk 6 and 7. Feeding SBO at 1.25% led to an increase (P < 0.0001) in the long-chain omega-3 polyunsaturated fatty acids (LCn3PUFA) and a concomitant decrease (P < 0.0001) in arachidonic acid (ARA) in the egg yolk lipids. Yet this amount of SBO in the diet had no effect (P > 0.1) on the sensory attributes of the egg and on production parameters such as egg weight, number of eggs laid, and feed intake (P > 0.05). When feeding SBO in amounts higher than 1.25% proportionately, a plateau effect of the LCn3PUFA content of the eggs was observed. This appears to be because the PUFA content in the sn-2 position of the phospholipids cannot exceed a certain amount. When this amount is reached, the LCn3PUFA will be increasingly stored in triglycerides. The results presented here clearly indicate how eggs can be produced with optimized composition of LCn3PUFA without affecting (P > 0.1) the sensory properties of the eggs. The procedures elaborated herein provide directly applicable consequences for the food industry.

Detection of potential markers for systemic disease in saliva of pigs by proteomics: a pilot study.

Animals with different health status have been studied in order to extend the knowledge about protein composition of porcine saliva samples and to discover potential salivary markers for systemic disease in porcine production. Clinical examination of animals was performed at farm level where 10 healthy pigs and 10 animals with evident clinical signs of disease were randomly selected. Saliva and blood samples were obtained and afterwards animals were humanely sacrificed to perform a complete necropsy. Levels of two acute phase proteins, haptoglobin and C-reactive protein, were used to identify possible active infections of the animals. Moreover, serological analysis, to the main porcine infectious diseases in the area, was performed. Salivary proteins were separated by two-dimensional gel electrophoresis followed by mass spectrometry for the identification of specific proteins. A total of 58 spots out of 75 were successfully identified by MS, which correspond to 20 unique proteins. Two different approaches were used to perform a statistical comparison of saliva protein patterns from healthy and diseased animals using the relative spot volume (% spot volume/total volume of all spot in the gel, approach "A") or taking also into account the total protein content of each saliva sample (µg of spot/mL of saliva, approach "B"). Both analyses showed three proteins in common that are differentially regulated between states. However, approach B was selected for biomarker searching since it gave an estimation of protein concentration and showed differential expression of proteins between both health states in a total of 10 proteins, which were up-regulated in disease. Mass spectrometric analysis identified those proteins as salivary lipocalin, lipocalin 1, double headed protease inhibitor protein, adenosine deaminase, haptoglobin, albumin fragments, S100-A8, S100-A9, S100-A12 and pancreatic alpha amylase. These proteins could be considered as potential salivary markers of disease.

A proteomic analysis of serum from dogs before and after a controlled weight-loss program.

The objective of this study was to investigate how weight-loss program would alter the proteome of the serum of Beagle dogs. For this purpose, serum samples from 5 Beagle dogs, before and after weight loss, were analyzed using 2-dimensional electrophoresis. Protein profiles of all samples were obtained, divided into 2 classes (obese and lean), and compared using specific 2-dimensional software, giving a total of 144 spot matches. Statistical analysis revealed 3 spot matches whose expressions were modulated in response to weight loss: 2 protein spots were upregulated and 1 protein spot was downregulated in the obese state in comparison with the lean state of the dogs. Mass spectrometric identification of differentially regulated spots revealed that these protein spots corresponded to retinol-binding protein 4, clusterin precursor, and α-1 antitrypsin, respectively, which could be considered potential markers of obesity and obesity-related disease processes in dogs.

Aflatoxin B(1) in affecting broiler's performance, immunity, and gastrointestinal tract: a review of history and contemporary issues.

Aflatoxin B(1) is a common contaminant of poultry feeds in tropical and subtropical climates. Research during the last five decades has well established the negative effects of the mycotoxin on health of poultry. However, the last ten years of relevant data have accentuated the potential of low levels of aflatoxin B(1) to deteriorate broiler performance. In this regard, any attempt to establish a dose-effect relationship between aflatoxin B(1) level and broiler performance is also complicated due to differences in types of broilers and length of exposure to the mycotoxin in different studies. Contrary to the prevalent notion regarding literature saturation with respect to aflatoxicosis of chicken, many areas of aflatoxicosis still need to be explored. Literature regarding effects of the mycotoxin on the gastrointestinal tract in this regard is particular scanty and non-conclusive. In addition to these issues, the metabolism of aflatoxin B(1) and recently proposed hypotheses regarding biphasic effects of the mycotoxin in broilers are briefly discussed.