Automated on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry method for the determination of ochratoxin A in wine and beer

An automated on-line solid-phase extraction-liquid chromatography-electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) method was developed for the determination of ochratoxin A (OTA) in alcoholic beverages. Mean recoveries for wine and beer were, respectively, 75 and 82%. Detection was achieved in negative ionization with a Q TRAP mass spectrometer operating in multiple-reaction monitoring (MRM) mode or enhanced product ion (EPI) mode, using the third quadrupole as linear ion trap. The MRM mode turned out to be more sensitive; the method allowed accurate determination of OTA in the range of 0.01-25 ng mL(-1) using external calibration. Within-day and between-day relative standard deviation percentages were <6.2 and <9.1%, respectively. In EPI mode, fragmentation spectra at the limit of quantification (0.03 ng mL(-1)) and good linearity could be obtained. Application of the method (MRM mode) to the analysis of several wine and beer samples purchased in local stores revealed OTA levels in the ranges of 0.03-1.44 ng mL(-1) for wines and 0.02-0.14 ng mL(-1) for beers.     

Simple assay for analyzing five microcystins and nodularin in fish muscle tissue: hot water extraction followed by liquid chromatography-tandem mass spectrometry

A simple, specific, and sensitive procedure for determining six cyanotoxins, that is, microcystins RR, LR, YR, LA, and LW and nodularin, in fish muscle tissue is presented. This method is based on the matrix solid-phase dispersion technique with heated water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissue by 4 mL of water acidified to pH 2 and heated at 80 degrees C. After acidification and filtration, 0.2 mL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, with at least two precursor ion > product ion transitions selected for each target compound. Analyte recovery ranged between 61 and 82% and was not substantially affected by either the analyte concentrations or the type of fish. The nonexcellent recovery of some of the microcystins was traced to binding of these compounds to protein phosphatases in fish tissue occurring during sample treatment. The existence of covalently bound microcystins in fish has been evidenced by several studies. Compared to an older sample preparation procedure, this one extracted larger amounts of the analytes in a simpler and much more rapid way. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 1.6 and 4.0 ng/g. The effects of temperature and volume of the extractant on the analyte recovery were studied.     

Near-infrared reflectance spectroscopy (NIRS) for protein, tryptophan, and lysine evaluation in quality protein maize (QPM) breeding programs

Quality protein maize (QPM) has approximately twice the tryptophan (Trp) and lysine (Lys) concentrations in protein compared to normal maize. Because several genetic systems control the protein quality of QPM, it is essential to regularly monitor Trp and/or Lys in breeding programs. Our objective was to examine the potential of near-infrared reflectance spectroscopy (NIRS) to enhance the efficiency of QPM research efforts by partially replacing more expensive and time-consuming wet chemistry analysis. More than 276 maize samples were used to develop NIRS models for protein content (PC), Trp, and Lys. The standard error of prediction (SEP) for the calibration and the coefficient of determination for validation (R(2)(v)) were 0.26 and 0.96 for PC, 0.005 and 0.85 for Trp, and 0.02 and 0.75 for Lys. When the NIRS models were used to evaluate 266 S2 lines from five QPM breeding populations, the coefficients of determination between NIRS and the chemical data were 0.94, 0.76, and 0.80 for PC, Trp, and Lys, respectively. Therefore, the NIRS models can be used to support the QPM breeding efforts.     

Quantification of transmission of foot-and-mouth disease virus caused by an environment contaminated with secretions and excretions from infected calves

Foot-and-mouth disease virus (FMDV) infected animals can contaminate the environment with their secretions and excretions. To quantify the contribution of a contaminated environment to the transmission of FMDV, this study used calves that were not vaccinated and calves that were vaccinated 1 week prior to inoculation with the virus in direct and indirect contact experiments. In direct contact experiments, contact calves were exposed to inoculated calves in the same room. In indirect contact experiments, contact calves were housed in rooms that previously had held inoculated calves for three days (either from 0 to 3 or from 3 to 6 days post inoculation). Secretions and excretions from all calves were tested for the presence of FMDV by virus isolation; the results were used to quantify FMDV transmission. This was done using a generalized linear model based on a 2 route (2R, i.e. direct contact and environment) SIR model that included information on FMDV survival in the environment. The study shows that roughly 44% of transmission occurs via the environment, as indicated by the reproduction ratio R^02Renvironment that equalled 2.0, whereas the sum of R^02Rcontact and R^02Renvironment equalled 4.6. Because vaccination 1 week prior to inoculation of the calves conferred protective immunity against FMDV infection, no transmission rate parameters could be estimated from the experiments with vaccinated calves. We conclude that a contaminated environment contributes considerably to the transmission of FMDV therefore that hygiene measures can play a crucial role in FMD control.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0156-5) contains supplementary material, which is available to authorized users.     

No foot-and-mouth disease virus transmission between individually housed calves

The foot-and-mouth disease outbreak in The Netherlands in 2001 most likely started on a mixed veal-calf/dairy-goat farm. The outbreak among the 74 calves on this farm appeared to be limited to four animals, and no clinical signs of FMD were reported. Also on a second veal-calf farm minor clinical signs and limited virus transmission were observed. Since FMD is known to be a very contagious disease, and can cause severe lesions, these observations were disputed. Therefore, we carried out two experiments to determine whether the Dutch FMD virus isolate from 2001 does spread among individually housed calves with limited contacts, either indirect (experiment 1) or direct (experiment 2). In experiment 1, four pairs of calves were housed in an individual box at 1m distance from each other. In experiment 2, two groups of three calves were housed in individual boxes, directly bordering each other. We infected one animal per pair in experiment 1, and the calf in the middle in experiment 2. We recorded clinical signs, virus shedding in saliva and the development of antibodies. In addition, we determined whether the virus was transmitted from the inoculated calves to the neighbour(s). All inoculated calves showed mild signs of FMD--fever, and some vesicles on hooves and/or in the mouth--but only one calf showed signs that were visible without physical examination. All inoculated calves shed virus in the saliva and developed neutralising antibodies. None of the contact animals seroconverted, indicating that virus transmission did not occur. These experiments showed that no virus transmission among individual housed calves can occur. This finding supports the hypothesis of the route of virus introduction to The Netherlands in 2001 and show that the observations on the two veal-calf farms were not impossible.     

Comparable sensitivity and specificity in three commercially available ELISAs to differentiate between cattle infected with or vaccinated against foot-and-mouth disease virus

Three commercially available ELISAs for the detection of antibodies to the non-structural proteins of foot-and-mouth disease virus (FMDV) were evaluated, using sera from uninfected, vaccinated, infected, inoculated, first vaccinated and subsequently infected, and first vaccinated and subsequently inoculated cattle. We compared antibody kinetics to non-structural proteins, sensitivity, and specificity. One of the ELISAs had a higher sensitivity and much lower specificity than the other two, therefore we established standardised cutoff values for the compared assays using receiver operated characteristic (ROC) curves. Using the standardised cutoff values, all three ELISAs produced comparable results with respect to sensitivity and specificity. Antibody development to non-structural proteins after infection and after vaccination/infection was not significantly different. Development of antibodies, however, both neutralising and directed to non-structural proteins, was significantly delayed after intranasal inoculation as compared to intradermolingual infection. Based on results of sera obtained after vaccination and experimental infection all three assays can be used for testing sera collected between 4 weeks and 6 months after infection. More information is needed on the prevalence of positive reactors in a situation where emergency vaccination has been used and FMD transmission was still observed.     

Serum Protein Fraction in Mature Horses and Relationship With Metabolic and Hematological Parameters

This study was conducted to determine the effect of age on serum protein fractions and their relationship with metabolic and hematological profiles in mature horses. Twenty-five mature Italian Saddle horses (mean age 13.6 ± 4.8 years) fed the same diet (grass hay and concentrate) were stratified according to age as first maturity, M1 (≤10 years old); second maturity, M2 (>10 and <15 years old); and old, O (>15 years), to be monitored every 28 days for a continuous period of 140 days. Horses in group O had higher plasma protein and thiol concentrations and white blood cell and neutrophil counts than the other two groups. Serum α₂-globulin concentrations were positively correlated with total plasma cholesterol (r = 0.514; P < .001), alkaline phosphatase (r = 0.430; P < .001), aspartate aminotransferase (r = 0.339; P < .001), ceruloplasmin (r = 0.321; P < .001), glutamic pyruvate transaminase (r = 0.444; P < .001), reactive oxygen metabolites (r = 0.426; P < .001), and blood neutrophil counts (r = 0.344; P < .01), and negatively with plasma bilirubin (r = −0.522; P < .001) and creatinine (r = −0.400; P < .001). These results suggest differences in hematological and metabolic profile in Italian Saddle horses after 15 years of age, resulting mainly from changes in plasma proteins and inflammatory mediators. The α₂-globulins fraction seems a quick but reliable marker of an inflammatory situation that, successively, should be better investigated with specific metabolites or enzymes.