This study describes quantitative analyses of some penicillins in goat milk after therapeutic treatment of bacterial infections. Group 1 (systemic infections, n = 5) had been treated intramuscularly with amoxicillin (AMX, 7 mg/kg b.w., 5x within 5 days), group 2 (mastitis; n = 6) intramammarily with oxacillin/ampicillin (OXA/AMP, 40/20 mg, 4x within 4 days) in one udder half. Udder half milk samples were analyzed by enzyme immunoassays, with detection limits of 1 ng/ml (AMX), 30 ng/ml (OXA), and 2 ng/ml (sum OXA/AMP). AMX residues varied between 1 and 160 ng/ml during treatment, but fell below 1 ng/ml 1 day before the end of the withdrawal period. In all samples except one, AMX concentrations were below the European Union maximum residue limit (MRL, 4 ng/ml) as early as two days after the last application. OXA levels in treated udder halves varied widely during treatment (<30 ng/ml to 1.2 mg/ml). Milk from non-treated halves also contained OXA, but concentrations were lower (<30 - 410 ng/ml). OXA fell below the MRL (30 ng/ml) 1.5 days before the end of the withdrawal period. Combined analysis of OXA and AMP showed that total residues were well below 4 ng/ml at least 1 day before the end of the withdrawal period. This is the first quantitative report on OXA excretion in goat milk after therapeutic application under real life conditions of goat milk production. The milk of all analysed goats complied with MRL regulations after the withdrawal period of 4 days (OXA) and 4.5 days (AMX).

This study investigated several food safety criteria in 38 different commercial products of processed cereal-based foods (PCF) from the German market. Microbiological assessment, followed by 16S RNA gene sequencing of suspect colonies, included aerobic mesophilic bacteria, moulds, Enterobacteriaceae, Cronobacter spp., and presumptive Bacillus cereus. Mycotoxin analyses were performed by enzyme immunoassays for deoxynivalenol (DON), zearalenone (ZEN), T-2/HT-2 toxins (T-2/HT-2; oat containing products only), ergot alkaloids (EA), and alternariol (AOH). No violative result above existing European Union regulations or international guidelines was obtained. Most samples had very low aerobic mesophilic cell counts (<2.0 × 101 CFU/g), the maximum was 9.6 × 102 CFU/g. A few samples contained low numbers of opportunistic pathogens, most notably Cronobacter sakazakii, Acinetobacter spp., Pantoea spp., and enterotoxigenic Bacillus wiedmannii. Levels of mycotoxin contamination were very low, well below European Union maximum limits. DON was found in 10 samples, at levels of 9-35 µg/kg. T-2/HT-2 were found in all 15 oat-based products (1-8 µg/kg). All samples were negative for ZEN and EA. A high number (n = 25) of samples yielded weakly positive results for the nonregulated AOH (0.4-2 µg/kg), but just three samples exceeded a level of 1 µg/kg. No relationship between cereal composition and analytical findings for microbiological parameters and mycotoxins could be found. As long as PCF meals are freshly prepared and consumed immediately after preparation, the risk from sporadically occurring opportunistic bacteria appears to be minimal.

The present study aimed to develop a novel enzyme immunoassay (EIA) for the tremorgenic mycotoxin, penitrem A (PTA). This competitive EIA, based on rabbit anti-PTA polyclonal antibodies and a PTA-bovine serum albumin conjugate, yielded a mean standard curve detection limit of 220 ng/mL. The PTA-EIA, together with two EIAs for paxilline (PAX) and ergoline alkaloids, were used to study the toxin profiles in culture of fungi isolated from some naturally infected food sample materials, including dairy products, citrus fruits, and walnuts. PTA was produced by eight out of 27 mycelium extracts from malt extract agar and Sabouraud glucose chloramphenicol selective agar (2-200 mu g/mL). Ten isolates from all types of food were also positive in the PAX-EIA (0.004-4 mu g/ mL). The EIA for ergoline alkaloids yielded positive results (0.0002-0.4 mu g/mL) in isolates from dairy products and from walnuts, but not from citrus fruits. Control analyses of selected fungal extracts by HPLC-MS/MS for PTA and PAX qualitatively confirmed the EIA results, poor quantitative agreement could be attributed to the presence of penitrems other than PTA, and to PAX analogues, respectively. Selected PTA-positive fungal isolates were subjected to sequencing of the internal transcribed spacer region and the beta-tubulin gene, and were identified as Penicillium polonicum and P. crustosum. In conclusion, Penicillium spp. capable to produce PTA, PAX, and ergoline alkaloids, either alone or in combination, appear to be quite common as contaminants on foods at the retail or production level. Some are capable to produce PTA in culture at high levels, which would have to be considered as toxic if present in food.

Zusammenfassung Gegenstand und Ziel Zum Ausscheidungsverhalten von Antibiotika in Milch nach therapeutischer Applikation bei klinisch erkrankten Ziegen liegen keine Daten vor. In dieser Studie wurden daher in 2Ziegenmilch-Betrieben in einem Zeitraum von 4Monaten Halftengemelksproben aller im Routinebetrieb klinisch erkrankter und daraufhin mit Antibiotika behandelter Tiere bis 3Tage nach Ablauf der Wartezeit auf Ruckstande untersucht. Material und Methoden Entsprechend den eingesetzten Praparate ergaben sich 3Behandlungsgruppen. Tiere der Gruppe1 (n=5; bakterielle Infektionen exklusive Mastitis) waren intramuskular mit Amoxicillin behandelt worden. Bei Tieren der Gruppe2 (n=6) lag eine akute katarrhalische Mastitis vor, die mit Oxacillin und Ampicillin intramammar behandelt worden war. Bei Gruppe3 handelte es sich um eine einzelne Ziege mit bakteriell bedingter Mastitis, die, nach erfolgloser Behandlung mit Oxacillin/Ampicillin, gema ss eines Antibiogramms Cefquinom intramammar erhalten hatte. Die Milchproben wurden vergleichend mit 2Testsytemen (Rezeptor-Schnelltest, Betastar ((R)) ; Brillantschwarz-Reduktionstest, BRT) qualitativ bzw. teilweise auch semiquantitativ auf Ruckstande untersucht. Ergebnisse Mittels BRT bzw. Betastar ((R)) konnten alle Wirkstoffe im Bereich der Ruckstandshochstmengen (Maximum Residue Limits, MRL) nachgewiesen werden. In Gruppe1 zeigten jeweils beide Euterhalften ahnliche Ausscheidungsverhaltnisse. Amoxicillin war meist nur 1Tag, maximal 3Tage nach Behandlungsende nachweisbar. In den Gruppen 2 und 3 (intramammare Applikation in eine Euterhalfte) zeigten sich erhebliche Konzentrationsunterschiede zwischen behandelter und unbehandelter Halfte, doch wiesen auch Milchproben der unbehandelten Euterhalften im Behandlungszeitraum Gehalte deutlich uber den MRLs auf. Ruckstande waren in beiden Gruppen maximal 4Tage nach Behandlungsende nachweisbar. Klinische Relevanz Es ergaben sich keine Hinweise auf eine verlangerte Ausscheidung der gepruften Antibiotika bei klinisch kranken Ziegen, die Wartezeiten waren zur Sicherstellung der Ruckstandsfreiheit ausreichend. Die eingesetzten Testsysteme erwiesen sich als gut geeignet zur Ruckstandskontrolle von Ziegenmilch nach therapeutischer Applikation. Lediglich bei stark verandertem Milchsekret konnten teilweise keine auswertbaren Ergebnisse erzielt werden. Abstract Objective Data on the excretion of antibiotic residues following therapeutic drug dosages in lactating goats with clinical signs of bacterial infections are currently lacking. Therefore, this study aimed at monitoring the drug residue excretion of a subset of frequently used antibiotics in the milk of dairy goats following their therapeutic administration. Material and methods Over a period of 4months, milk samples (udder halves) were collected in 2goat milk farms from animals treated with antibiotics in routine practice based on the diagnosis of a bacterial infection. The samples were examined up to 3days following the withdrawal time point. The animals were classified in 3groups depending on their clinical symptoms and treatment. Goats in group1 (afebrile goats with various bacterial infections excluding the udder) were treated with intramuscular amoxicillin injection (n=5). Animals in group2 (mastitis catarrhalis) were treated with intramammary injection of oxacillin and ampicillin (n=6). Group3 consisted of a single goat diagnosed with mastitis. This individual was treated with cefquinome in accordance with the results of the antibiogram. Milk samples were examined qualitatively by using a receptor assay (Betastar ((R)) ) as well as a microbiological inhibitor assay (Brilliant black reduction test, BRT). The latter assay was also used to semiquanti-tatively analyse drug residue levels in samples from group2. Results Following intramuscular treatment with amoxicillin, drug residue levels were estimated to be very similar in both udder halfs. Elimination was complete 3days after the end of the treatment period. Animals in group2 showed significant differences between treated and untreated udder halves. However, the untreated halves still exhibited residue levels exceeding the maximum residue limits during the treatment period. In both group 2 and 3, all milk samples were tested negative for drug residues before the end of the withdrawal period. Conclusion In the present study, no evidence of prolonged residue excretion into milk of goats following therapeutic administration of antibiotics was detected. Both the receptor test and the BRT represent suitable methods for an efficient antibiotic drug residue testing in goat milk. Reliable testing was merely not attainable in cases of milk samples possessing highly altered organoleptic characteristics.

A novel nucleic acid lateral flow immunoassay (NALFIA) system for Cronobacter (C.) sakazakii. and C. malonaticus is based on amplification of the rpoB gene in the presence of a heterobifunctional, Cronobacter-specific DNA probe. Partial degradation of this probe during amplification is then detected by lateral flow immunoassay. The NALFIA detected all of the 22 C. sakazakii and 8 C. malonaticus isolates under study, but showed no reaction with other Cronobacter species or non-Cronobacter species. The minimal visual and instrumental detection limits were 104 cfu/ml for both species in buffered peptone water and in reconstituted infant formula. C. sakazakii or C. malonaticus at levels of 10(0)-10(1) cfu/g in powdered infant formula were reliably detected by NALFI after reconstitution and 24 h incubation at 37 degrees C. A major advantage of this approach is that the use of an integrated DNA probe provides additional test specificity. Since C. sakazakii and C. malonaticus are by far the most important Cronobacter species occurring in PIF, the novel NALFIA is a promising tool to enhance rapid control of infant formula.

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, α-ergocryptine, and ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

A newly developed enzyme immunoassay (EIA) for the detection of the tremorgenic indole-diterpene alkaloid paxilline (PAX) and closely related analogs was used to analyze ergot sclerotia collected from rye and barley fields. The mean EIA standard curve detection limit was 0.47 ± 0.14 ng/mL; relative cross-reactivity of toxin standard solutions was found for 11-hydroxy-paspaline (terpendole E, 1.1%) but not for lolitrem B or ergot alkaloids. Sclerotia from all fields were positive in the PAX-EIA at concentration levels of 620 ± 200 and 160 ± 37 μg/kg in ergot of rye and 130 ± 47 μg/kg in ergot of barley. Confirmatory analyses of sclerotia by liquid chromatography-tandem mass spectrometric detection identified PAX and its analog 13-desoxypaxilline. To the best of our knowledge, this is the first report on the natural occurrence of tremorgenic indole-diterpene alkaloid mycotoxins in ergot sclerotia from rye and barley. Along with details on the analytical methodology developed in this study, particularly PAX-antibody production, the relevance and implications of these findings for food and feed safety are discussed. Presence or absence of elevated levels of tremorgenic mycotoxins, along with the ergot alkaloids, would help in explaining the difference between the two distinct manifestations of historic ergotism, the convulsive and the gangrenous form. Further method development for paxilline and other tremorgenic mycotoxins in cereals used for food and feed is a prerequisite for a comprehensive risk assessment, which seems to be necessary in light of the findings reported here.

Limited availability of toxin standards for lolitrem B and ergovaline impedes routine control of grasses for endophyte toxins. This study aimed at assessing the applicability of an enzyme immunoassay (EIA) for the indole-diterpene mycotoxin paxilline, in combination with a generic EIA for ergot alkaloids, as alternative parameters for screening purposes. Analysis of grass seeds and model pastures of four different grass species showed that both EIAs yielded highly positive results for paxilline and ergot alkaloids in perennial ryegrass seeds. Furthermore, evidence for natural occurrence of paxilline in grass in Germany was obtained. High performance liquid chromatography-tandem mass spectrometry analysis qualitatively confirmed the paxilline EIA results but showed that paxilline analogues 1'-O-acetylpaxilline and 13-desoxypaxilline were the predominant compounds in seeds and grass. In the absence of easily accessible reference standards for specific analysis of some major endophyte toxins, analysis of paxilline and ergot alkaloids by EIA may be suitable substitute parameters. The major advantage of this approach is its ease of use and speed, providing an analytical tool which could enhance routine screening for endophyte toxins in pasture.

A total of 63 brined cheeses (30 white pickled cheeses, 33 Tulum cheeses) were randomly collected from informal market places such as weekly street markets in the province Ankara during a seven-month period, and quantitatively analysed for Staphylococcus (S.) aureus. S. aureus isolates obtained from these samples were analysed for staphylococcal enterotoxin (SE) genes by PCR, selected isolates were tested for SEA-SEE production by enzyme immunoassay. S. aureus was found in eleven (17.0 %) of cheese samples at levels between 9.5 x 10(2) and 5.0 x 10(6) cfu/g. All 22 S. aureus isolates were positive by PCR for one or more toxin genes (sea, sed, seg, sei, selj, sem, sen, seo, sep, ser and selu), forming three distinct toxin profiles. SEA or SED production was found for isolates from five samples, by enzyme immunoassay. Further characterization of isolates by macrorestriction analysis yielded three different pulsed-field gel electrophoresis (PFGE) profiles which corresponded well with SE gene profiles. Identical PFGE profiles were obtained for isolates from several alleged unique cheeses, purchased from different vendors in different markets, indicating a common source of production and disproving the claimed originality. These findings highlight the existence of health hazards related to consumption of traditional cheeses originating from such underregulated markets and the need to implement more intensive hygiene control measures.

The tremorgenic paxilline (PAX), an indole-diterpene alkaloid mycotoxin, was recently detected as a natural contaminant of ergot of barley and rye. To check the possibility of a transfer of this mycotoxin into beer, a rapid and sensitive immunochemical method for the analysis of PAX in commercial bottled beer was developed. A straightforward sample preparation procedure could be established, including degassing, pH adjustment, optional filtering, and finally a 1:5 dilution with a methanolic phosphate-buffered saline solution. Analysis of PAX was performed by a competitive indirect enzyme immunoassay (EIA). The detection limit at a cut-off value of 80% B/B0 of the EIA standard curve was evaluated by analysis of spiked beer. PAX at levels of 3 μg/L and 5 μg/L yielded 69% and 100% positive results, therefore the detection limit in beer was at 5 μg/L. Recoveries of PAX at levels of 5–20 μg/L were 88–97%, coefficients of variation were 17–22%. With these characteristics, the EIA was considered to be a suitable screening method for PAX in beer. A survey of bottled beer from the German market was conducted which included 38 samples of domestic and international brands, the latter containing various flavoring ingredients. All samples were clearly negative for PAX, which indicates that this toxin is not a relevant contaminant in beer.

Cronobacter spp. cause infant disease, several cases have been associated with powdered infant formulae (PIF). In the early 2000s, contamination of German PIF with these opportunistic pathogens was quite common. Before 2008, all isolates Cronobacter spp. had been classified as Enterobacter sakazakii, therefore little is known about species diversity within such isolates. Genetic, serologic, and biochemical traits of 80 Cronobacter isolates, originally obtained 2003-2006 within infant food surveys in Germany, were reassessed in this study. By sequencing of the fusA gene, all isolates were unambiguously assigned to two species, C sakazakii (n = 73) and C. malonaticus (n = 7). PCR serotyping identified five C. sakazakii serotypes and two C. malonaticus serotypes, biochemical profiling yielded five biogroups. PFGE analysis also showed high heterogeneity in both species. Multilocus sequence typing of 26 selected isolates yielded 16 different sequence types (ST), including C sakazakii ST 1 (n = 6) and the highly virulent ST 4 (n = 2). The results suggest that just two, but highly heterogeneous species were responsible for the Cronobacter contamination problem which challenged the German PIF industry in the beginning of this century. This fact may have influenced the success of efforts to identify and eliminate sources of contamination. (C) 2017 Elsevier Ltd. All rights reserved.

Objective: To determine the occurrence of CTX-M producing Escherichia coli (E. coli) from cattle feces in Bogor slaughterhouse, Indonesia.Methods: A total of 220 cattle feces samples were collected from Bogor slaughterhouse from March to April 2015. Presence of extended-spectrumbeta-lactamase (ESBL) producing E. coli was detected by disc diffusion test based on the recommendation from Clinical and Laboratory Standards Institute (2014). Bacterial strains which were confirmed as producing ESBLs were further analyzed for the presence of bla genes of the ESBL by PCR.Results: The results showed that CTX-M producing E. coli isolates were detected in 19 samples from 220 samples (8.6%). The b-lactamase genes detected were CTX-M-1 (n = 10) and CTX-M-9 (n = 9). All of the CTX-M producing E. coli isolates showed multidrug resistance phenotypes to at least four antibiotics. The highest incidence of antibiotics resistance was showed to ampicillin (100.0%), cefotaxime (100.0%), and cefpodoxime (100.0%), followed by streptomycin (84.3%), trimethoprim-sulfamethoxazole (73.7%), erythromycin (52.6%), kanamycin (26.3%), doxycycline (10.5%), and ceftazidime (0.0%).Conclusions: Detection of CTX-M-producing E. coli in cattle feces raises important questions as they can represent a potential risk factor to public health.

A simplified method to produce specific polyclonal rabbit antibodies against sterigmatocystin (STC) was established, using a STC-glycolic acid-ether derivative (STC-GE) conjugated to keyhole limpet haemocyanin (immunogen). The competitive direct enzyme immunoassay (EIA) established for STC had a detection limit (20% binding inhibition) of 130 pg ml(-1). The test was highly specific for STC, with minor cross-reactivity with O-methylsterigmatocystin (OMSTC, 0.87%) and negligible reactivity with aflatoxins (< 0.02%). STC-EIA was used in combination with a previously developed specific EIA for aflatoxins (< 0.1% cross-reactivity with STC and OMSTC), to study the STC/aflatoxin production profiles of reference strains of Aspergillus species. This immunochemotaxonomic procedure was found to be a convenient tool to identify STC- or aflatoxin-producing strains.

Although the dairy farm environment is a known source of extended-spectrum β-lactamase (ESBL)-producing bacteria, surveillance data on ESBL in the milk production chain are still scarce. This study aimed at estimating the dimensions of the problem for public health and animal welfare by surveying ESBL-producing Enterobacteriaceae in raw bulk tank milk in Germany. Samples from 866 dairy farms, comprising about 1% of the total number of dairy farms in Germany, were first screened for presence of cefotaxime-resistant bacteria by selective enrichment. Suspect colonies were identified phenotypically and further characterized by biochemical and molecular methods, including analysis of resistance genes and clonal diversity in ESBL-producing isolates. Bulk tank milk from 82 (9.5%) farms yielded Enterobacteriaceae with confirmed ESBL-production. The most frequent ESBL-producing species was Escherichia coli (75.6%), followed by Citrobacter spp. (9.6%), Enterobacter cloacae (6.1%), and Klebsiella oxytoca (3.7%), a few isolates belonged to other species within the genera Hafnia, Raoutella and Serratia. The majority of isolates (95.1%) harbored the β-lactamase blaCTX-M gene, which has gained increased importance among ESBL-producing strains worldwide; the CTX-M group 1 was found to be the dominating (88.4%) phylogenetic group. All ESBL-positive Escherichia coli isolates were clonally heterogeneous, as determined by pulsed-field gel electrophoresis. The results from this survey demonstrate that ESBL-producing bacteria are distributed widely in the dairy farm environment in Germany. Therefore, raw milk is a potential source of exposure for the consumer, which is of increasing importance considering the trend of farmer-to-consumer direct marketing. Furthermore, dairy farm staff have an increased likelihood of exposure to ESBL-producing bacteria. Finally, ESBL-producing bacteria may also be transferred via waste milk to calves, thus further spreading antibiotic resistance in the farm environment.