Pasteurella sp. associated with fatal septicaemia in six African elephants.

The sudden mortality of African elephants (Loxodonta africana) in Botswana and Zimbabwe in 2020 provoked considerable public interest and speculation. Poaching and malicious poisoning were excluded early on in the investigation. Other potential causes included environmental intoxication, infectious diseases, and increased habitat stress due to ongoing drought. Here we show evidence of the mortalities in Zimbabwe as fatal septicaemia associated with Bisgaard taxon 45, an unnamed close relative of Pasteurella multocida. We analyse elephant carcasses and environmental samples, and fail to find evidence of cyanobacterial or other intoxication. Post-mortem and histological findings suggest a bacterial septicaemia similar to haemorrhagic septicaemia caused by P. multocida. Biochemical tests and 16S rDNA analysis of six samples and genomic analysis of one sample confirm the presence of Bisgaard taxon 45. The genome sequence contains many of the canonical P. multocida virulence factors associated with a range of human and animal diseases, including the pmHAS gene for hyaluronidase associated with bovine haemorrhagic septicaemia. Our results demonstrate that Bisgaard taxon 45 is associated with a generalised, lethal infection and that African elephants are susceptible to opportunistically pathogenic Pasteurella species. This represents an important conservation concern for elephants in the largest remaining metapopulation of this endangered species.

A qualitative peptide biomarker approach to identify piscine gelatine in products to support food security.

Due to allergy concerns, it is mandatory under EU law to declare in food products all ingredients derived from fish. Gelatine is prepared from the waste collagen of animal carcasses, including piscine, bovine and porcine materials, and is an ingredient in a wide range of foods. The Elliott Review into the integrity and assurance of food supply networks highlighted requirements for analytical surveillance methods to support due diligence, food safety and authenticity. We present the development of a method to extract gelatine from foods and determine the presence of piscine gelatine by liquid chromatography-tandem mass spectrometry using a suite of sixteen piscine marker peptides. The method has been successfully applied to gelatine granules, capsules and composite retail food products. While a study is required to determine parameters including the limit of detection of this method, the data indicate the method is reproducible between replicates of sub-samples and applies to a range of piscine gelatines collected over 16 years. Once validation studies are complete, there is potential for enforcement officers to apply the technology to verify the authenticity of fish products to support consumers in ensuring food safety and also food provenance relating to animal origin.

The determination of monomers and oligomers from polyester-based can coatings into foodstuffs over extended storage periods.

The polymeric coating used in metal packaging such as cans for foods and beverages may contain residual amounts of monomers used in the production of the coating, as well as unreacted linear and cyclic oligomers. Traditionally, although designed for use with plastic food contact materials, food simulants have been used to determine the migration of monomers from coatings into foodstuffs. More recently, food simulants have also been used to determine oligomeric species migrating from can coatings. In the work reported here, the migration of both monomers and oligomers from polyester-based can coatings into food simulants and foodstuffs, some of which were towards the end of their shelf-life, is compared. The concentrations of monomers and selected oligomers in canned foods at the end of their shelf life were found to be significantly lower than those in food simulants, which in turn was lower than those in the extraction solvent acetonitrile.

Occurrence of polybrominated diphenylethers, hexabromocyclododecanes, bromophenols and tetrabromobisphenols A and S in Irish foods.

The occurrence of polybrominated diphenyl ethers (PBDEs), hexabromocyclododecanes (HBCDs), tetrabromobisphenol A (TBBPA) and other phenolic brominated flame retardants (BFRs) in Irish foodstuffs has been assessed. A total of 53 food samples including eggs, milk, fish, fat and offal were tested. Eighty-one percent of the samples contained at least one measurable PBDE congener. The most abundant and frequently occurring congeners were BDE-47, BDE-49, BDE-99, BDE-100 and BDE-209 with the highest concentrations found in fish, fat and eggs. Summed concentrations for the measured PBDEs ranged from 0.02 µg/kg to 1.37 µg/kg whole weight. At least one HBCD stereoisomer was found in twenty-six percent of the samples with α-HBCD being the most frequently detected. The highest concentrations were found in fat and oily fish samples. TBBPA was only detected in one farmed salmon sample at 0.01 µg/kg. Bromophenol residues were found in fourteen out of the 53 samples, specifically in eggs and fish, with concentrations ranging from 0.28 to 0.98 µg/kg whole weight. These data contribute to the EU-wide EFSA risk assessment on these contaminants that is currently underway.

Evaluation of direct analysis in real time for the determination of highly polar pesticides in lettuce and celery using modified Quick Polar Pesticides Extraction method.

Direct analysis in real time (DART) was evaluated for the determination of a number of highly polar pesticides using the Quick Polar Pesticides Extraction (QuPPe) method. DART was hyphenated to high resolution mass spectrometry (HRMS) in order to get the required selectivity that allows the determination of these compounds in complex samples such as lettuce and celery. Experimental parameters such as desorption temperature, scanning speed, and distances between the DART ion source and MS inlet were optimized. Two different mass analyzers (Orbitrap and QTOF) and two accessories for sample introduction (Dip-it® tips and QuickStrip™ sample cards) were evaluated. An extra clean-up step using primary-secondary amine (PSA) was included in the QuPPe method to improve sensitivity. The main limitation found was in-source fragmentation, nevertheless QuPPe-DART-HRMS proved to be a fast and reliable tool with quantitative capabilities for at least seven compounds: amitrole, cyromazine, propamocarb, melamine, diethanolamine, triethanolamine and 1,2,4-triazole. The limits of detection ranged from 20 to 60µg/kg. Recoveries for fortified samples ranged from 71 to 115%, with relative standard deviations <18%.

Masked trichothecene and zearalenone mycotoxins withstand digestion and absorption in the upper GI tract but are efficiently hydrolyzed by human gut microbiota in vitro.

SCOPE: Cereal grains are commonly contaminated with Fusarium mycotoxins and their plant-derived masked metabolites. The fate of masked mycotoxins in the human gut is poorly understood. Here we assess the metabolism and transport of glucoside metabolites of common trichothecenes (deoxynivalenol, nivalenol, T-2 toxin) and zearalenone compounds (zearalenone, α- and ß-zearalenol) in the human gut in vitro. METHODS AND RESULTS: Masked mycotoxins were incubated with artificial digestive juices and absorption was assessed in differentiated Caco-2/TC7 cells. Colonic metabolism was studied using fecal batch cultures from five donors and mycotoxins were detected using LC-MS/MS. All masked mycotoxins were stable under upper GI tract conditions and no absorption was observed. Free trichothecenes were absorbed intact whereas free zearalenone compounds were absorbed and metabolized to undetected compounds by Caco-2/TC7 cells. Human gut microbiota efficiently hydrolyzed all masked mycotoxins. Trichothecenes were fully recovered as parent mycotoxins whereas 40-70% of zearalenone compounds were further metabolized to unknown metabolites. CONCLUSION: Our results demonstrate that masked trichothecenes will reach the colon intact to be released as parent mycotoxins by gut microbiota, hence contributing to mycotoxin exposure. Masked zearalenone compounds are metabolized by gut microbiota and epithelial cells and the identity and toxicity of metabolites remain to be determined.

Use of atmospheric pressure solids analysis probe time-of-flight mass spectrometry to screen for plasticisers in gaskets used in contact with foods.

RATIONALE: Plasticisers are used in the PVC gaskets of metal closures on glass jars and bottles used for foods and beverages. They may migrate and so contaminate the packed foodstuff. The plasticisers are present in a high proportion and are often a complex mixture of substances leading to time-consuming analytical methodologies. This work describes a rapid screening method to identify the plasticisers used. METHODS: Analysis was carried out by direct sampling of the gaskets using atmospheric pressure solids analysis probe (ASAP) with time-of-flight (TOF) mass spectrometry (MS) using a SYNAPT G2 HDMS system. The accurate mass information collected was then compared to a user-prepared database of plasticisers to aid identification. RESULTS: The rapid identification approach was shown to be successful for 24 gasket samples previously analysed by alternative more lengthy gas chromatographic (GC) methods. Quantification by dissolution followed by standard addition was also demonstrated to be reliable. CONCLUSIONS: The ASAP-TOFMS method is a useful technique for rapidly screening gaskets for the presence of plasticisers. It can be used to identify specific gaskets deserving of further quantitative analysis by chromatographic methods, saving time and money by avoiding unnecessary analyses. Copyright © 2015 John Wiley & Sons, Ltd.

Development of a food compositional database for the estimation of dietary intake of phyto-oestrogens in a group of postmenopausal women previously treated for breast cancer and validation with urinary excretion.

The scientific literature contains evidence suggesting that women who have been treated for breast cancer may, as a result of their diagnosis, increase their phyto-oestrogen (PE) intake. In the present paper, we describe the creation of a dietary analysis database (based on Dietplan6) for the determination of dietary intakes of specific PE (daidzein, genistein, glycitein, formononetin, biochanin A, coumestrol, matairesinol and secoisolariciresinol), in a group of women previously diagnosed and treated for postmenopausal breast cancer. The design of the database, data evaluation criteria, literature data entry for 551 foods and primary analysis by LC­MS/MS of an additional thirty-four foods for which there were no published data are described. The dietary intake of 316 women previously treated for postmenopausal breast cancer informed the identification of potential food and beverage sources of PE and the bespoke dietary analysis database was created to, ultimately, quantify their PE intake. In order that PE exposure could be comprehensively described, fifty-four of the 316 subjects completed a 24 h urine collection, and their urinary excretion results allowed for the description of exposure to include those identified as 'equol producers'.

Volatile fingerprints of seeds of four species indicate the involvement of alcoholic fermentation, lipid peroxidation, and Maillard reactions in seed deterioration during ageing and desiccation stress.

The volatile compounds released by orthodox (desiccation-tolerant) seeds during ageing can be analysed using gas chromatography-mass spectrometry (GC-MS). Comparison of three legume species (Pisum sativum, Lathyrus pratensis, and Cytisus scoparius) during artificial ageing at 60% relative humidity and 50 °C revealed variation in the seed volatile fingerprint between species, although in all species the overall volatile concentration increased with storage period, and changes could be detected prior to the onset of viability loss. The volatile compounds are proposed to derive from three main sources: alcoholic fermentation, lipid peroxidation, and Maillard reactions. Lipid peroxidation was confirmed in P. sativum seeds through analysis of malondialdehyde and 4-hydroxynonenal. Volatile production by ageing orthodox seeds was compared with that of recalcitrant (desiccation-sensitive) seeds of Quercus robur during desiccation. Many of the volatiles were common to both ageing orthodox seeds and desiccating recalcitrant seeds, with alcoholic fermentation forming the major source of volatiles. Finally, comparison was made between two methods of analysis; the first used a Tenax adsorbent to trap volatiles, whilst the second used solid phase microextraction to extract volatiles from the headspace of vials containing powdered seeds. Solid phase microextraction was found to be more sensitive, detecting a far greater number of compounds. Seed volatile analysis provides a non-invasive means of characterizing the processes involved in seed deterioration, and potentially identifying volatile marker compounds for the diagnosis of seed viability loss.

Progress towards the characterisation of faecal compounds.

Intestinal nitrosation produces ATNCs (Apparent Total N-nitroso Compounds) and these have been linked with an increased risk of colon cancer from eating red meat. Modern LC-MS instrumentation makes direct detection of ATNC components in faecal water a possibility. The difficulty is in determining which of the many compounds present are N-nitrosamines before embarking on efforts to characterise them. We have assumed that any in vivo nitrosation of alimentary tract contents will be non-specific and depend on the amount and basicity of amine present, with concentration of nitrosating agent being the limiting factor. By further nitrosating faecal waters (and ileostomy fluids) we can increase the amount of ATNC and readily access these compounds. The amount and the number of nitrosamines generated depend on the concentration of individual amines present. By derivatisation separately using both 14N and 15N labelled nitrite, we demonstrated that inspecting chromatograms in parallel for a unit mass difference provides a novel and practicable means for identifying unknown ATNC in faecal and ileostomy samples. MS procedures were linked with the traditional approaches of thermal energy analyser (TEA), preparative HPLC, visualisation of nitrosamines with Griess reagent and degradation of N-nitroso compounds by UV irradiation. We have demonstrated that this approach is repeatable and have used it to identify 30 putative N-nitroso compounds (as protonated parent masses [M + H]+ at: 242, 258, 312, 313, 333, 348, 365, 377, 382, 386, 392, 412, 414, 421, 434, 442, 466, 467, 483, 493, 572, 582, 625, 636, 637, 656, 662, 752, 808, and 870.

Mass spectral studies towards more reliable measurement of perfluorooctanesulfonic acid and other perfluorinated chemicals (PFCs) in food matrices using liquid chromatography/tandem mass spectrometry.

Liquid chromatography/mass spectrometry (LC/MS) experiments are described, leading to a reliable method for the measurement of perfluorooctanesulfonic acid (PFOS) and other perfluorinated chemicals (PFCs) in foods. Separations were performed on new fluorinated stationary phases, RP Octyl (-C(8)F(17)) or propyl-perfluorobenzene (-C(3)H(6)-C(6)F(5)), to ensure resolution of PFOS and interfering taurohydroxycholate isomers. Aqueous ammonium formate (5 mM) and methanol were used as the mobile phases. The mass spectrometer was operated in negative electrospray ionisation mode, recording two transitions for each analyte and one for each internal standard. The purities of the analytical standards for the eleven target perfluoro analytes (C(7) to C(12) carboxylic acids, C(4), C(6) and C(8) sulfonic acids, and octanesulfonamide (PFOSA)) were found to be in close agreement with the supplied values; the lowest purity was 91%. Five candidate internal standards were investigated, (13)C(4)-PFOS, (13)C(4)-perfluorooctanoic acid, (13)C(2)-perfluorodecanoic acid, D(9)-n-ethylperfluorooctanesulfonamidoethanol (D(9)-n-Et-FOSE) and D(3)-n-methylperfluorooctanesulfonamide (D(3)-n-Me-FOSA); the purities were all >98%. The use of tetrahydro-PFOS generated backgrounds (>1 microg/kg) for perfluoroheptanoic acid and perfluorobutanesulfonic acid. Similarly D(9)-n-Et-FOSE was unacceptable and D(3)-n-Me-FOSA was volatile, leaving no clear candidate for normalisation of the measurement of PFOSA. Severe matrix-induced suppression and enhancement effects influenced ionisation, making external calibration and quantification problematic. This was addressed by a parallel standard addition and matrix-matching approach, comparing ionisation in methanol, in procedural blanks and in food-based extracts. The limits of detection (LODs) of 0.001-0.01 microg/kg in solvent and 0.01-1 microg/kg in foods demonstrate that this method is suitable for the determination of PFCs in all food to the required 1 microg/kg reporting level.

Acrylamide analysis: assessment of results from six rounds of Food Analysis Performance Assessment Scheme (FAPAS) proficiency testing.

Six proficiency tests have now been completed in an ongoing program of the UK Food Analysis Performance Assessment Scheme (FAPAS) for the analysis of acrylamide in a range of food matrixes. Homogeneous test material samples were requested by laboratories throughout the world, with 29 to 45 submitting results for each test. Results were analyzed by appropriate statistical procedures, and z-scores were awarded for reported values. In the absence of both legislation and collaborative trial data, the target standard deviation was derived from the Horwitz equation, although it is acknowledged that there is a need to establish a "fit for purpose" target standard deviation specifically for acrylamide analysis. Participants were encouraged to use the analytical method routinely used in their own laboratory and to provide details of their procedure. Close examination of the data submitted indicates that performance is generally acceptable in terms of accuracy. There is no significant difference between results submitted by gas chromatography and liquid chromatography (GC and LC) methods, and no method dependency on the use of internal standards or sample size. However, choice of extraction solvent may be important, with indications that plain water is an acceptable extraction method. There is evidence from the most recent test that direct (underivatized) GC methodology may present problems, but more data are required and this aspect will be monitored in the continuing proficiency testing program.

Influence of 10 wk of soy consumption on plasma concentrations and excretion of isoflavonoids and on gut microflora metabolism in healthy adults.

BACKGROUND: Little information is currently available on the role of the gut microflora in modulating isoflavone bioavailability or on sex differences in isoflavone metabolism and bioavailability. OBJECTIVE: We sought to determine whether chronic soy consumption influences isoflavone bioavailability as judged by plasma isoflavone concentrations and modified gut microflora activities [beta-glucoside hydrolysis and equol and O-desmethylangolensin (O-DMA) production]. We also examined whether sex differences in isoflavone metabolism exist. DESIGN: A randomized, parallel, controlled study design was used to compare a high-soy diet (104 +/- 24 mg total isoflavones/d) with a low-soy diet (0.54 +/- 0.58 mg total isoflavones/d) in 76 healthy young adults for 10 wk. RESULTS: Concentrations of isoflavones and their gut microflora metabolites in the plasma, urine, and feces were significantly higher in the subjects who consumed the high-soy diet than in those who consumed the low-soy diet. Concentrations of O-DMA in plasma and urine were higher in the men than in the women. Fecal bacteria from subjects consuming both diets could convert daidzein to equol ex vivo. Fecal beta-glucosidase activity was significantly higher in the subjects who consumed the high-soy diet than in those who consumed the low-soy diet. CONCLUSIONS: Although interindividual variation in isoflavone metabolism was high, intraindividual variation was low. Only concentrations of O-DMA in plasma and urine appeared to be influenced by sex. Chronic soy consumption does not appear to induce many significant changes to the gut metabolism of isoflavones other than higher beta-glucosidase activity.

Influence of soya-based infant formula consumption on isoflavone and gut microflora metabolite concentrations in urine and on faecal microflora composition and metabolic activity in infants and children.

The urinary excretion of soya isoflavones and gut microflora metabolites was investigated in infants and children who had been fed soya-based infant formulas in early infancy. These infants and children were compared with cows'-milk formula-fed controls, to determine at what age gut microflora metabolism of daidzein to equol and/or O-desmethylangolensin (O-DMA) was established, and whether exposure to isoflavones in early infancy influences their metabolism at a later stage of development. Sixty infants and children (aged 4 months-7 years) participated in the study; thirty in each of the soya and control groups. There were four age groups. These were: 4-6 months (seven in the soya group and seven in the control group); 7-12 months (seven in the soya group and nine in the control group); 1-3 years (six in the soya group and eight in the control group); 3-7 years (ten in the soya group and six in the control group). Urine samples were collected to measure isoflavonoids by MS, and faecal samples were collected to measure gut-health-related bacterial composition, by fluorescent in situ hybridisation with oligonucleotide probes, and metabolic activity. A soya challenge (typically a soya yoghurt alternative product containing 4.8 g soya protein and on average 22 mg total isoflavones) was given to control-group infants (>6 months) and children, and also to soya-group children that were no longer consuming soya, to determine their ability to produce equol and/or O-DMA. Urinary genistein, daidzein and glycitein were detected in all infants (4-6 months) fed soya-based infant formula; O-DMA was detected in 75 % of infants but equol was detected in only 25 %. In the controls (4-6 months), urinary isoflavonoids were very low or not detected. In the older age groups (7 months-7 years), O-DMA was found in the urine samples of 75 % of the soya group and 50 % of the controls, after the soya challenge. Equol excretion was detected in 19 % of the soya-group infants and children, and in only 5 % of the controls. However, in the oldest (3-7 years) children, the proportion excreting O-DMA and equol was similar in both groups. Faecal bacterial numbers for bifidobacteria (P<0.001), bacteroides and clostridia (P<0.05) were significantly lower for the soya group compared with the control group. There appears to be no lasting effect of early-life isoflavone exposure on isoflavone metabolism.

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards.

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.