•This review discusses immunoassays developed for the detection of macrocyclic lactones in food matrices.•Hapten synthesis, bioconjugation, and antibody production for macrocyclic lactones are reviewed.•Advantages and limitations of different types of immunoassays are summarized.•Current rapid sample preparation methods and commercial tests are summarized and discussed.Macrocyclic lactones including the avermectins, macrolides, and milbemycins are a group of antimicrobial agents that have been widely used in food-producing animals, and residues from these drugs may be present in edible tissues, milk, and eggs for human consumption. Thus, easy, rapid, and sensitive methods are required for the effective screening of these drugs in food and related matrices. Antibodies have been used as analytical tools in various assays and analysis techniques developed for food safety because of their inherent specificity and high affinity. This review discusses the advances in antibody-based analytical methods, i.e., immunoassays, for the screening and detection of macrocyclic lactones in food matrices. Hapten synthesis, bioconjugation, and antibody production for macrocyclic lactones are reviewed. Current sample preparation methodologies and the academic and commercial immunoassay tests for residues of macrocyclic lactones in food matrices are summarized and discussed.

•Comparison of prevalence of erm(B) in Campylobacter isolates amongst three representative regions and between the three years.•Novel study of erm(B) in Campylobacter conducted in constant regions within a defined time period.A total of 1372 Campylobacter isolates (1107 Campylobacter coli and 265 Campylobacter jejuni) were obtained from 3462 samples collected from slaughterhouses and farms in three representative regions of China (Shandong, Guangdong, and Shanghai) over three successive years (2013–2015). Of these, 84 (84/1372, 6.1%) were erm(B)-positive, and all 84 positive isolates were identified as C. coli (83 chicken isolates and one swine isolate). The prevalence of erm(B) in Campylobacter isolates was compared amongst the different regions and between the three years investigated. The rates of erm(B)-positive Campylobacter in Guangdong increased remarkably over the experimental period (3.8% to 22.8%), while their higher rates observed in Shanghai (4.4%) and Shandong (2.4%) occurred in 2015 and 2014. Further, 72 erm(B)-positive isolates were associated with the type V and VI multidrug-resistance genomic islands (MDRGIs), which have previously only been identified in human Campylobacter isolates, while one isolate of chicken origin contained the type II MDRGI, which has previously been detected in swine isolates. Expansion of the erm(B) in Campylobacter with similar PFGE and MLST type from chicken isolates from Shanghai and Guangdong to human isolates identified previously in Shanghai was also observed. The findings in this study confirmed previously observed trend of dissemination of erm(B) and MDRGIs in zoonotic Campylobacter isolates and provide new insights into the prevalence of erm(B)-positive Campylobacter isolates in chickens and swine from three representative regions of China over a consecutive 3-year period.

Here, we describe a general bioluminescence resonance energy transfer (BRET) homogeneous immunoassay based on quantum dots (QDs) as the acceptor and Renilla luciferase (Rluc) as the donor (QD-BRET) for the determination of small molecules. The ratio of the donor–acceptor that could produce energy transfer varied in the presence of different concentrations of free enrofloxacin (ENR), an important small molecule in food safety. The calculated Förster distance (R0) was 7.86 nm. Under optimized conditions, the half-maximal inhibitory concentration (IC50) for ENR was less than 1 ng/mL and the linear range covered 4 orders of magnitude (0.023 to 25.60 ng/mL). The cross-reactivities (CRs) of seven representative fluoroquinolones (FQs) were similar to the data obtained by an enzyme-linked immunosorbent assay (ELISA). The average intra- and interassay recoveries from spiked milk of were 79.8–118.0%, and the relative standard deviations (RSDs) were less than 10%, meeting the requirement of residue detection, which was a satisfactory result. Furthermore, we compared the influence of different luciferase substrates on the performance of the assay. Considering sensitivity and stability, coelenterazine-h was the most appropriate substrate. The results from this study will enable better-informed decisions on the choice of Rluc substrate for QD-BRET systems. For the future, the QD-BRET immunosensor could easily be extended to other small molecules and thus represents a versatile strategy in food safety, the environment, clinical diagnosis, and other fields.

Campylobacter jejuni, an important foodborne microorganism, poses severe and emergent threats to human health as antibiotic resistance becomes increasingly prevalent. The mechanisms of drug resistance are hard to decipher, and little is known at the metabolic level. Here we apply metabolomic profiling to discover metabolic changes associated with amphenicol (chloramphenicol and florfenicol) resistance mutations of Campylobacter jejuni. An optimized sample preparation method was combined with ultra-high-performance liquid chromatography–time-of-flight mass spectrometry (UHPLC–TOF/MS) and pattern recognition for the analysis of small-molecule biomarkers of drug resistance. UHPLC–triple quadrupole MS operated in multiple reaction monitoring mode was used for quantitative analysis of metabolic features from UHPLC–TOF/MS profiling. Up to 41 differential metabolites involved in glycerophospholipid metabolism, sphingolipid metabolism, and fatty acid metabolism were observed in a chloramphenicol-resistant mutant strain of Campylobacter jejuni. A panel of 40 features was identified in florfenicol-resistant mutants, demonstrating changes in glycerophospholipid metabolism, sphingolipid metabolism, and tryptophan metabolism. This study shows that the UHPLC–MS-based metabolomics platform is a promising and valuable tool to generate new insights into the drug-resistant mechanism of Campylobacter jejuni.

•The effect of hapten incorporation on the titer and sensitivity of antisera was studied.•The specificity of monoclonal antibody was evaluated using 17 macrolide antibiotics.•A ciELISA for the determination of erythromycin in milk was developed.Erythromycin is an antibiotic used extensively in veterinary practice worldwide for treatment, prevention and growth promotion. In this work, monoclonal antibodies (Mabs) against erythromycin were produced and used to develop a competitive indirect enzyme-linked immunosorbent assay (ciELISA) for the determination of erythromycin in milk. A novel carboxyphenyl derivative of erythromycin (ERO-CMO) was synthesized and conjugated with bovine serum (BSA) for use as the immunogen or ovalbumin (OVA) as the coating antigen. Four hybridoma cell lines were isolated, which produced Mabs that competed with erythromycin. The 6C1 and 5B2 Mabs had IC50 values for erythromycin of 14.40 and 0.94 μg L−1, respectively. These Mabs demonstrated high cross-reactivity to the macrolides containing 14-membered rings, but not to oleandomycin. No cross-reactivity was observed for 12 macrolides that contained 15 or 16-membered lactone rings or for 2 pleuromutilins. The ciELISA developed using the 5B2 Mab afforded recovery values that ranged from 76.9% to 85.7% with only a 10-fold sample dilution prior to analysis.

A fluorescence-based immunochromatographic assay (ICA) for fumonisin B1 (FB1) that employs conjugates of fluorescent microspheres and monoclonal antibodies (FM–mAbs) as detection reporters is described. The ICA is based on the competitive reaction between FB1-bovine serum albumin (BSA; test line) and the target FB1 for binding to the FM–mAb conjugates. A limit of detection (LOD) for FB1 of 0.12 ng/mL was obtained, with an analytical working range of 0.25–2.0 ng/mL (corresponding to 250–2000 μg/kg in maize flour samples, according to the extraction procedure). The recoveries of the ICA to detect FB1 in maize samples ranged from 91.4 to 118.2%. A quantitative comparison of the fluorescence-based ICA and HPLC-MS/MS analysis of naturally contaminated maize samples indicated good agreement between the two methods (r2 = 0.93). By replacing the target of interest, the FM-based ICA can easily be extended to other chemical contaminants and thus represents a versatile strategy for food safety analysis.

Chloramphenicol (CAP) is a forbidden antibiotic that enters the food chain by illegal use in food-producing animals, potentially causing aplastic anaemia in humans. Immuno-PCR has the potential to address the need of meeting strict limits for this antibiotic by detecting trace levels of CAP present in animal-derived foods. A real-time immuno-quantitative PCR (RT-IPCR) assay for the quantification of CAP based on the simple and quick immunomagnetic bead recovery of CAP in milk was developed. The immunomagnetic beads were obtained by linking the reporter DNA to anti-CAP monoclonal antibody with N-succinimidyl S-acetylthioacetate (SATA) and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and immobilising the anti-CAP monoclonal antibody/DNA-label conjugate on the magnetic beads with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (SNHS). The RT-IPCR assay allows the sensitive detection and quantification of CAP in the concentration range of 0.001–0.11 μg L−1 (R2 = 0.9986) with a 50% inhibition concentration (IC50) value of 0.008 μg L−1. The RT-IPCR approach discussed here is presented as a model system that could be easily adapted for small molecule detection in a variety of foods using simple immunomagnetic bead recovery.

•A new derivative of ractopamine was synthesized and used as a ligand.•Several antibody purification procedures were conducted and compared.•The newly designed affinity chromatography method provided the highest recovery.The application of antibodies to small molecules in the field of bioanalytics requires antibodies with stable biological activity and high purity; thus, there is a growing interest in developing rapid, inexpensive and effective procedures to obtain such antibodies. In this work, a ractopamine (RAC) derivative, N-4-aminobutyl ractopamine (ABR), was synthesized for preparing new specific affinity chromatography to purify a murine monoclonal antibody (mAb) against RAC from ascites. The performance of the new specific chromatography was compared with four other purification methods in terms of recovery, purity and biological activity of mAb. These four purification methods were prepared by using specific ligands (RAC and RAC-ovalbumin) and commercial ligands (protein G and protein A), respectively. The results showed that the highest recovery (88.1%) was achieved using the new chromatography; in comparison, the recoveries from the other methods were all below 70%. The purity of the mAbs from the new chromatography was 88.3%, while, the highest purity of 97.6% was from protein G chromatography and the lowest purity of 84.7% was from protein A chromatography. The biological activity of the purified mAb from all of the chromatography methods was comparable in enzyme-linked immunosorbent immunoassay (ELISA).

This paper describes the preparation of reusable immunoaffinity columns and the development of an ultra-performance liquid chromatography tandem mass spectrometry method combined with immunoaffinity column clean-up (IAC-UPLC-MS/MS) for the determination of ochratoxin A (OTA) in cereals and feeds. The monoclonal antibody (mAb) was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1 (κ-light chain) isotype. A competitive indirect enzyme-linked immunosorbent assay was used to characterize the mAb. The concentrations causing 50 % inhibition of binding of mAb to OTA-ovalbumin by free OTA, ochratoxin B, and ochratoxin C were 1.29, 4.78, and 0.94 ng mL−1, respectively. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N >10) ranging from 0.5 to 1.0 μg kg−1 and a limit of detection (LOD, S/N >3) ranging from 0.2 to 0.3 μg kg−1 in cereal and feed samples. The IAC-UPLC-MS/MS method offers a good LOQ and LOD for OTA in cereal and feed samples. The accuracy and precision at this level fall within the EU regulatory limit. This methodology has been validated in four different matrices (millet, maize, soybean, and swine finisher diet) with highly satisfactory results and applied to the analysis of samples collected from the markets.

Enzyme-linked immunosorbent assays (ELISAs) usually focus on the detection of a single analyte or a single group of analytes, e.g., fluoroquinolones or sulfonamides. However, it is often necessary to simultaneously monitor two classes of antimicrobial residues in different food matrixes. In this paper, we describe a dual-colorimetric ELISA for the simultaneous detection of 13 fluoroquinolone and 22 sulfonamide residues. The limit of detection for fluoroquinolones and sulfonamides was 2.4 and 5.8 ng/mL, respectively. The developed immunoassay is suitable for high-throughput screening of these low-molecular weight contaminants. This is the first report where two different enzymes (alkaline phosphatase and horseradish peroxidase) were used in one immunoassay and together in a single well for simultaneous detection of multiple low-molecular weight chemical residues.

This paper presents a sensitive and confirmatory multi-residue method for the analysis of 23 veterinary drugs and metabolites belonging to three classes (nitroimidazoles, benzimidazoles, and chloramphenicols) in porcine muscle, liver, and kidney. After extracted with ethyl acetate and basic ethyl acetate sequentially, the crude extracts were defatted with hexane and further purified using Oasis MCX solid-phase extraction cartridges. Rapid determination was carried out by ultra-high performance liquid chromatography–electrospray ionization tandem mass spectrometry. Data acquisition was performed under positive and negative mode simultaneously. Recoveries based on matrix-matched calibrations for meat, liver, and kidney ranged from 50.6 to 108.1%. The method quantification limits were in the range of 3–100 ng/kg.Highlights► A reliable method for determination of three classes of drugs in swine tissues. ► The sensitivity of the method can be obtained at ng/kg level (ppt). ► Electrospray ionization was performed at positive and negative mode simultaneously.

Immunoassays contribute greatly to food safety. Yet there are no reported immunoassays that simultaneously detect MQCA and QCA, the marker residues for olaquindox and carbadox, respectively. Here, a broad-specificity mAb was successfully produced, and the mAb showed good cross-reactivity with both MQCA and QCA, having IC50 values in buffer of 4.8 and 9.6 ng/mL, respectively. The calibration curves ranged from 0.3 to 81 μg/kg. The average recoveries ranged from 76% to 108% at different spiked levels (2, 4, and 8 μg/kg for MQCA; and 4, 10, and 20 μg/kg for QCA), and the intra-/interday coefficients of variation were 4.2–13.3%. The limits of detection of MQCA and QCA in chicken, fish, pork, and shrimp were 1.76, 1.32, 1.90, and 1.18 μg/kg, respectively. This method was verified by LC–MS/MS, with a correlation coefficient above 0.98. The immunoassay was rapid and reliable for simultaneous screening analysis of MQCA and QCA residues.

Three immunizing haptens, 6-(4-aminophenylsulfonamido)hexanoic acid (HS), 4-(4-(4-aminophenylsulfonamido)phenylsulfonamido)benzoic acid (SSS) and 4-(4-(4-aminophenylsulfonamido)phenyl)butanoic acid (SA10), were synthesized and used as haptens to produce generic polyclonal antibodies against sulfonamides. A novel generic enzyme-linked immunosorbent immunoassay (ELISA) for the detection of sulfonamides was developed based on one site heterologous coating hapten derived from sulfadiazine (STZ), providing IC50 values below 100 ng mL−1 for the 26 sulfonamides tested in buffer. The study showed that the site heterologous coating hapten could significantly improve the sensitivity of ELISA in comparison to a homologous or bridge length heterologous coating hapten. The developed ELISA was used first for the detection of five sulfonamides in spiked chicken muscle with recovery values ranging from 80.9 to 87.4% when the clean-up step was not included and from 85.6% to 92.7% when the clean-up step was included in sample pretreatment. The study showed that the high sensitivity and broad specificity of the assay make it a suitable screening method for the determination of multi-sulfonamides residues in chicken muscle.

Aflatoxins are a class of highly toxic chemical contaminants occurring in food. Consumption of aflatoxin-contaminated food can lead to harmful effects on human health. A rapid and reliable analysis of aflatoxins in food is crucial. In this study, we generated a broad–specific monoclonal antibody (MAb) 3 C10 against aflatoxin B1 (AFB1). The MAb 3 C10 binds specifically to AFB1 and AFM1 and has a IC50 of 0.13 μg L−1 for AFB1 and 0.16 μg L−1 for AFM1. Furthermore, the MAb showed high cross-reactivity to AFB2, AFG1, and AFG2. To enable simultaneous AFB1 and AFM1 detection in different food matrices, an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on MAb 3 C10 has been developed and optimized. In addition, the extraction methods of different food matrices (peanut, corn, soybean, wheat flour, rice, soy sauce, vinegar, wine, raw milk, pure milk, skimmed milk, and yogurt) were established. The average recovery ranged from 73 to 121 %, with relative standard deviation values less than 15 %. The limit of detection was 0.52 + 0.36 μg kg−1 (mean + 3SD) for AFB1 in eight agricultural products and 0.031 + 0.015 μg kg−1 (mean + 3SD) for AFM1 in four dairy products. The sensitivity of icELISA was below the limit set by the European Commission for aflatoxin detection in different food matrices and similar to LC–MS/MS method. We demonstrate a rapid, simple, and reliable method for simultaneous screening of AFB1 and AFM1 in different food matrices.

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC50) at concentrations below 100 ng mL–1. MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC50 values ranging from 1.2 to 12.4 ng mL–1, almost within 1 order of magnitude. These produced MAbs show lower IC50 values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies.

A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC50 value of the method was 0.153 μg kg−1 for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA–F–BSA (FFA–formaldehyde–BSA) and FF–G–OVA (FF–glutaric anhydride–OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40:1 ethyl acetate–ammonia mixture, obtaining recoveries of 70.3–100% (FFA) and 71.8–102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCβ) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 μg kg−1 for FFA and 0.526 μg kg−1 for FF (10-fold dilution) and 0.453 μg kg−1 for FFA and 0.657 μg kg−1 for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCβ values of 1.0 μg kg−1 for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.

A sensitive and reliable method was developed and validated for the analysis of ractopamine in swine tissues. The sample preparation procedure was based on hydrolysis overnight, extraction with ethyl acetate, and solid-phase extraction cleanup. The target compound was determined by ultra-performance liquid chromatography coupled with tandem mass spectrometry. The average recoveries ranged between 93.3% and 106.5%, with relative standard deviations of 7.2–12.8%. The detection and quantification limits were 0.2 and 0.5 μg/kg, respectively. The depletion profile of ractopamine was studied in healthy pigs after oral administration of feed containing 20 mg/kg ractopamine for 30 consecutive days. Ractopamine residues were still detected 11 days post-medication in all tissues examined with the exception of muscle. The highest ractopamine level was found in lung tissue. The developed method has been successfully applied to the depletion study of ractopamine in swine tissues.Research highlights► Rapid UPLC-MS/MS analysis of ractopamine was performed in 4 min. ► The highest residual concentration of ractopamine was found in lung tissue. ► The estimated withdrawal time was 5 days.

A rapid and sensitive liquid chromatography–electrospray ionization-tandem mass spectrometry (LC–ESI-MS/MS) method to quantify thiamphenicol (TAP), florfenicol (FF), and florfenicol amine (FFA) in swine muscle is described. An immunoaffinity chromatography (IAC) column based on polyclonal antibodies and protein A-sepharose CL 4B was used to clean-up extracted samples. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of TAP, FF, and FFA. The dynamic column capacity was more than 512 ng/mL of gel after being used for 15 cycles. From fortified swine muscle samples at levels of 0.4–50 ng/g, the average recoveries were 85.2–98.9% with intra- and inter-day variations less than 9.8% and 12.4%, respectively. The limit of quantitation ranged from 0.4 to 4.0 μg/kg.

A method based on ultra-high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (UHPLC–MS/MS) for the simultaneous determination of benzimidazole residues in bovine milk has been optimized and validated. Rapid chromatographic separation of 13 analytes in 8 min was obtained by means of UHPLC. The samples were subject to Oasis MCX solid-phase extraction cartridges for extraction and clean-up. Matrix-matched calibration curves were performed to compensate for the matrix effect and loss in sample preparation. Mean recoveries ranged from 80% to 101% and inter-day precision was lower than 14%. Limit of detection and limit of quantification of the method ranged from 0.01 to 0.5 μg L−1 and from 0.1 to 1.0 μg L−1, respectively.

A fluorescence polarization immunoassay (FPIA) for the determination of salinomycin (SAL) was developed by using anti-SAL monoclonal antibodies (mAb). Fluorescein labeled SAL (tracer) was synthesized by the N-hydroxysuccinimide active ester method and purified using thin layer chromatography (TLC). The developed FPIA for SAL had a dynamic range from 0.60 to 2193 ng/mL with an IC50 value of 33.2 ng/mL and a detection limit (LOD) of 0.08 ng/mL. No significant cross-reactivities were observed with other drugs but 67.6% with narasin.

A liquid chromatographic method was developed for determination of nosiheptide in swine kidney and liver. The tissue samples were extracted with acetonitrile and defatted with hexane. The analytes were determined at a fluorescence excitation/emission wavelength of 357/515 nm and with gradient elution program. The limits of detection and limits of quantification were 5 and 20 ng g−1, respectively, for swine kidney and liver. The mean recoveries for nosiheptide in swine kidney and liver ranged from 70.3 to 97.4% with coefficients of variation below 11.3% at 20–100 ng g−1 fortification levels.

A sensitive and reliable method was developed and validated for detection and confirmation of melamine in egg based on gas chromatography–mass spectrometry (GC–MS) and ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS). Trichloroacetic acid solution was used for sample extraction and precipitation of proteins. The aqueous extracts were subjected to solid-phase extraction by mixed-mode reversed-phase/strong cation-exchange cartridges. Using ultra-performance liquid chromatography and electrospray ionization in the positive ion mode, melamine was determined by LC–MS/MS, which was completed in 5 min for each injection. For the GC–MS analysis, extracted melamine was derivatized with N,O-bis(trimethylsilyl)trifluoracetamide prior to selected ion monitoring detection in electron impact mode. The average recovery of melamine from fortified samples ranged from 85.2% to 103.2%, with coefficients of variation lower than 12%. The limit of detection obtained by GC–MS and UPLC–MS/MS was 10 and 5 μg kg−1, respectively. This validated method was successfully applied to the determination of melamine in real samples from market.

A rapid, sensitive and reliable multi-residue method for the simultaneous determination of four 5-nitroimidazoles (NIIMs) and their three corresponding metabolites in swine kidney was developed and validated. The compounds of interest were extracted from tissues with ethyl acetate. The crude extracts were subject to liquid–liquid partition with hexane followed by solid-phase extraction using mixed-mode strong cation-exchange column. Chromatographic separation was achieved on an AcQuity BEH C18 column and was completed within 4 min for each injection. Data acquisition under positive electrospray tandem mass spectrometry was performed by applying multiple reaction monitoring for both identification and quantification. Mean relative recoveries from fortified samples ranged from 83% to 111%, with coefficients of variation lower than 12%. The limits of detection and quantification for the NIIMs were in the range of 0.05–0.5 and 0.1–0.5 μg kg−1, respectively.

This work reports a rapid, reliable and sensitive multi-residue method for the simultaneous determination of six resorcylic acid lactones in bovine milk by ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). The resorcylic acid lactones were extracted, purified, and concentrated from milk samples in one step using a solid-phase extraction (SPE) cartridge that contained a polymeric mixed-mode anion-exchange sorbent. The analysis was performed on a Waters Acquity BEH C18 column utilizing a gradient elution profile. Each LC run was completed in 3.5 min. The analytes were detected by multiple reaction monitoring (MRM) using electrospray ionization (ESI) negative mode. Mean recoveries from fortified samples ranged from 92.6% to 112.5%, with relative standard deviations lower than 11.4%. Using 5 mL bovine milk, the limits of detection and quantification for resorcylic acid lactones were in the ranges of 0.01–0.05 and 0.05–0.2 μg/L, respectively. The application of this newly developed method was demonstrated by analyzing bovine milk samples from markets.

An immunoaffinity chromatographic method was developed using an antibody mediated immunosorbent to selectively extract and purify 10 quinolones (marbofloxacin, norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid, and flumequine) in chicken muscle followed by HPLC. The operating conditions of the immunoaffinity chromatography (IAC) column were optimized, and the IAC has been successfully used for the isolation and purification of 10 quinolones from chicken muscle tissue. The optimized immunoaffinity column sample cleanup procedure combined with HPLC coupling to fluorescence detection afforded low limits of detection (0.1 ng g−1 for danfloxacin and 0.15 ng g−1 for all other quinolones tested). The method was also applied to determine quinolone residues in commercial muscle samples.

A homogenous complement-mediated liposome immune lysis assay (LILA) was developed for the determination of enrofloxacin (ENRO) in carp and chicken muscle. ENRO was covalently coupled to DPPE, and then immobilized onto the surface of liposomes by reverse-phase evaporation method. The performed liposome would be specifically lysed by the sequential additions of anti-ENRO monoclonal antibody (MAb) and guinea pig complement. Through a competitive assay format, the performed liposome can be used to detect ENRO in a range of 5.0–20 ng mL−1 in assay buffer. The limit of detection of ENRO in carp and chicken muscle was 1 ng g−1 and the limit of quantification was 2 ng g−1. Recoveries ranged from 58.3% to 65.2% for carp and 55.6–63.8% for chicken muscle at spiked levels of 2–8 ng g−1, with intra-assay and inter-assay variations 5.6–12.3% and 7.1–19.2%, respectively.

This paper describes a novel mixed-bed immunoaffinity column (IAC) method. The IAC was produced by coupling anti-quinolone and anti-sulfonamide broad-specificity monoclonal antibodies to Sepharose 4B for simultaneously isolating 13 quinolones (QNs) and 6 sulfonamides (SAs) from swine and chicken muscle tissues, followed by antibiotic determination using liquid chromatography–tandem mass spectrometry (LC–MS/MS). A new broad-specificity Mab (B1A4E8) toward sulfonamides was produced using sulfamethoxazole as hapten that demonstrated cross-reactivities to 6 SAs in the range of 31–112%. IAC optimized conditions were found that allowed the IAC to be reused for selective binding of both SAs and QNs. Recoveries of all 19 antibiotics from animal muscle ranged from 72.6 to 107.6%, with RSDs below 11.3% and 15.4% for intra-day and inter-day experiments, respectively. The limit of quantification ranged from 0.5 to 3.0 ng/g. The strategy used here for a mixed-bed IAC may be used to study other compounds and more than two classes of analytes simultaneously.

A rapid and sensitive colloidal gold immunochromatography test strip based on one monoclonal antibody with broad-specificity, which can detect 12 fluoroquinolones (FQs), was developed. Antigen and goat anti-mouse IgG were respectively drawn on NC membrane as test line and control line. Gold-labeled antibody was added on a pad and put on one end of the membrane. Fluoroquinolones in sample solution compete with antigen combined on NC membrane for the gold-labeled antibody. When enough fluoroquinolone exists, the test line vanishes as there are no spare gold-labeled antibodies that can bind with antigen on the membrane. The control line always exists when the antibody is activated. The lowest detection limits of the FQs in spiked chicken muscle and chicken liver samples were 25 ng mL−1 for norfloxacin and pefloxacin. The lowest detection limit for the other 10 FQs (enrofloxacin, ciprofloxacin, norfloxacin, flumequine, pefloxacin, ofloxacin, lomefloxacin, enoxacin, danofloxacin, amifloxacin, oxolinic acid, and marbofloxacin) was 50 ng mL−1. The whole process involving sample preparation and detection can be finished in <10 min. The results demonstrate that the developed method can be potentially used as a screening tool for the determination of 12 FQ residues in a large amount of samples on site.

A method has been developed for simultaneous analysis of salinomycin and narasin in chicken muscle. Muscle samples were extracted with acetonitrile. Clean-up of the extracts on an immunoaffinity chromatography column was followed by liquid chromatography with postcolumn derivatisation and UV–visible detection at 520 nm. The immunoaffinity columns were prepared by coupling the anti-salinomycin monoclonal antibody to CNBr-activated Sepharose 4B. When chicken muscle fortified at 5, 25, and 50 ng g−1 was analyzed, intra-assay mean recoveries of salinomycin and narasin were in the ranges 87.5–93.1 and 86.2–94.3%, respectively, with relative standard deviation (RSD) of 4.7–6.2 and 2.4–5.7%. Inter-assay mean recoveries were 86.0–93.0 and 86.0–92.1%, respectively, with RSD of 4.8–6.5 and 5.8–7.4%. The limit of detection of the method was 2.5 ng g−1 for both drugs in chicken muscle.

A simple, rapid and sensitive liquid chromatographic method with programmable fluorescence and ultraviolet detection was developed and validated for simultaneous determination of seven fluoroquinolones (marbofloxacin, ofloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin and difloxacin) and four sulfonamides (sulfadiazine, sulfapyridine, sulfathiazole and sulfadimidine) in chicken muscle in a single run. The tissue sample was extracted with phosphate buffer (pH 6.0) and cleaned-up with a solid phase extraction cartridge. The mean recoveries for each drug in chicken muscle ranged from 78.0 to 105.2% with a relative standard deviation below 9.3% at 0.2–400 ng g−1 fortification levels. The limit of quantification was 0.2–4.0 ng g−1 for fluoroquinolones and 15.0 ng g−1 for sulfonamides.

A sensitive and reliable multiresidue method is described for analysis of ronidazole, metronidazole, dimetridazole and the common metabolite of ronidazole and dimetridazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole in swine liver. The sample preparation procedure was based on liquid–liquid extraction and mixed mode cation exchange/reverse phase solid-phase extraction. The compounds of interest were determined by reverse phase gradient liquid chromatography separation and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring (MRM) mode. The limits of confirmation were 0.1–0.5 μg kg−1 for the analytes.

A high-performance liquid chromatography (HPLC) method with ultraviolet (UV) detection was developed for the detection of cefquinome (CEQ) residues in swine tissues. The limit of detection (LOD) of the method was 5 ng g−1 for muscle and 10 ng g−1 for fat, liver, and kidney. Mean recoveries of CEQ in all fortified samples at a concentration range of 20–500 ng g−1 were 80.5−86.0% with coefficient of variation (CV) below 10.3%. Residue depletion study of CEQ in swine was conducted after five intramuscular injections at a dose of 2 mg kg−1 of body weight with 24 h intervals. CEQ residue concentrations were detected in muscle, fat, liver, and kidney using the HPLC-UV method at 265 nm. The highest CEQ concentration was measured in kidney tissue during the study period, indicating that kidney was the target tissue for CEQ. CEQ concentrations in all examined tissues were below the accepted maximum residue limit (MRL) recommended by the Committee for Veterinary Medical Products of European Medical Evaluation Agency (EMEA) at 3 days post-treatment.

A simple multiresidue method, HPLC with programmable fluorescence detection and gradient elution, has been developed for analysis of nine (fluoro)quinolones (FQs)—norfloxacin, ciprofloxacin, lomefloxacin, danofloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid, and flumequine in chicken muscle. The samples were extracted with phosphate-buffered saline (PBS, 0.01 mol L−1, pH 7.0) and cleaned by SPE. Cleaned extracts reconstituted in different solvents were tested to determine which gave the maximum fluorescence response for each drug. PBS (0.01 mol L−1, pH 7.0) was used as reconstitution solvent, because the sensitivity for FQs dissolved in PBS was 1.8–3.2 times greater than when dissolved in the mobile phase. Under the optimum conditions excellent linearity was obtained, with satisfactory correlation coefficients (r > 0.9994) for PBS. The “matrix effect” was eliminated. Limits of quantification for each drug were in the range 0.3–1.0 ng g−1. In fortification studies recoveries of the analytes were in the range 71.8–102.1% for 1–100 ng g−1 concentrations. Inter- and intra-day coefficients of variation were from 0.5 to 5.2% and from 1.7 to 9.0%, respectively. Short-term stability in PBS was also determined.

A simple multi-residue analysis method for the quantitative determination of eprinomectin, abamectin, doramectin and ivermectin in bovine tissues was developed. The tissue sample was extracted with acetonitrile, followed by clean-up on a C18 solid phase extraction cartridge. The eluate was derivatised before being analyzed by HPLC coupled to a fluorescence detector. The method was validated using bovine liver and muscle fortified with the drugs at 0, 5, 10 and 50 ng g−1. The mean recoveries of the four drugs were 70.31–87.11% in liver and 79.57–93.65% in muscle, with relative standard deviations below 17.84% in liver and 14.68% in muscle. The limits of detection were between 0.5 and 1.0 ng g−1 and the limits of quantification were 1–2 ng g−1 in bovine tissues for the four drugs.

Eprinomectin is a novel and potent antiparasitic animal health drug. An analytical procedure for the determination of EPR in bovine urine and feces has been developed. The urine sample was centrifuged and alkalized with ammonia following solid phase extraction. The fecal sample was extracted with acetonitrile, defatted with hexane, cleaned-up using C18 cartridge. All samples were analyzed by high performance liquid chromatography with fluorescence detection after derivatization with N-methylimidazole. The limits of detection are 0.5 ng mL−1 and 0.5 ng g−1, respectively. Fortified at 2, 10, 50, and 100 ng mL−1(ng g−1), inter-assay recoveries of EPR in cattle urine and feces were in the range of 87.9–91.5% and 78.6–86.3%, with coefficients of variation of 5.4–10.2% and 1.4–7.2%, respectively. Intra-assay mean recoveries of the analytes were 82.2–86.5% and 79.6–87.3%, with coefficients of variation of 7.8–11.5% and 6.3–7.8%, respectively. The method was used to study the excretion of eprinomectin in bovine urine and feces after subcutaneous administration at a dose of 0.5 mg kg−1.

The multidrug-resistance gene cfr was detected in coagulase-negative staphylococci from 3/401 pigs, 15/305 chickens, and 3/78 ducks from 31 different farms and one slaughterhouse in 2 provinces of China. Twenty of the 21 cfr-positive isolates were methicillin-resistant. Various SmaI-PFGE patterns were observed among the isolates of the same staphylococcal species and suggested horizontal transfer of cfr rather than clonal expansion. The detection of cfr on plasmids in the majority of the isolates supported this assumption. Analysis of the drug usage records of the farms strongly correlated with the resistance patterns of the cfr-positive isolates and suggested that antimicrobial use on farms supports the spread of the cfr gene.

BackgroundUntil now, polymyxin resistance has involved chromosomal mutations but has never been reported via horizontal gene transfer. During a routine surveillance project on antimicrobial resistance in commensal Escherichia coli from food animals in China, a major increase of colistin resistance was observed. When an E coli strain, SHP45, possessing colistin resistance that could be transferred to another strain, was isolated from a pig, we conducted further analysis of possible plasmid-mediated polymyxin resistance. Herein, we report the emergence of the first plasmid-mediated polymyxin resistance mechanism, MCR-1, in Enterobacteriaceae.MethodsThe mcr-1 gene in E coli strain SHP45 was identified by whole plasmid sequencing and subcloning. MCR-1 mechanistic studies were done with sequence comparisons, homology modelling, and electrospray ionisation mass spectrometry. The prevalence of mcr-1 was investigated in E coli and Klebsiella pneumoniae strains collected from five provinces between April, 2011, and November, 2014. The ability of MCR-1 to confer polymyxin resistance in vivo was examined in a murine thigh model.FindingsPolymyxin resistance was shown to be singularly due to the plasmid-mediated mcr-1 gene. The plasmid carrying mcr-1 was mobilised to an E coli recipient at a frequency of 10−1 to 10−3 cells per recipient cell by conjugation, and maintained in K pneumoniae and Pseudomonas aeruginosa. In an in-vivo model, production of MCR-1 negated the efficacy of colistin. MCR-1 is a member of the phosphoethanolamine transferase enzyme family, with expression in E coli resulting in the addition of phosphoethanolamine to lipid A. We observed mcr-1 carriage in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection.InterpretationThe emergence of MCR-1 heralds the breach of the last group of antibiotics, polymyxins, by plasmid-mediated resistance. Although currently confined to China, MCR-1 is likely to emulate other global resistance mechanisms such as NDM-1. Our findings emphasise the urgent need for coordinated global action in the fight against pan-drug-resistant Gram-negative bacteria.FundingMinistry of Science and Technology of China, National Natural Science Foundation of China.

•Antibiotic resistance is a complex global health challenge•One Health approaches are needed to facilitate understanding and action•China is one of the largest producers and consumers of antibiotics in the world•China is giving attention to antibiotic resistance at the highest political level•We report on the early stages of a Sino-Swedish One Health research programmeAntibiotic resistance is a complex global health challenge. The recent Global Action Plan on antimicrobial resistance highlights the importance of adopting One Health approaches that can cross traditional disciplinary boundaries. We report on the early experiences of a multisectoral Sino-Swedish research project that aims to address gaps in our current knowledge and seeks to improve the situation through system-wide interventions. Our research project is investigating antibiotic use and resistance in a rural area of China through a combination of epidemiological, health systems and laboratory investigations. We reflect here on the challenges inherent in conducting long distance cross-disciplinary collaborations, having now completed data and sample collection for a baseline situation analysis. In particular, we recognise the importance of investing in aspects such as effective communication, shared conceptual frameworks and leadership. We suggest that our experiences will be instructive to others planning to develop similar international One Health collaborations.

The present study aimed to identify, determine the susceptibility, and detect gene cassettes of Arcanobacterium (Actinomyces) pyogenes isolates from cows with endometritis. Arcanobacterium pyogenes isolates were identified first by using the API Coryne Vit system test, and further through PCR. Minimum inhibitory concentrations of 23 antimicrobial agents against A. pyogenes were tested using standard broth microdilution assays according to the protocols of the Clinical and Laboratory Standards Institute. The genes of integrons I and II were amplified by PCR using specific primers. Thirty-two A. pyogenes isolates were isolated from 136 endometritic cows in the Hohhot region. Antibiotic susceptibility tests revealed that all isolates were highly sensitive to fluoroquinolones (100%), macrolides (∼81.2 to 100%) and florfenicol (90.6%), aminoglycosides (∼15.6 to 81.2%), and tetracyclines (∼43.7 to 68.7%). However, 53.1% were resistant to clindamycin, ∼50 to 65.6% were resistant to penicillins, and ∼37.5 to 71.9% were resistant to cephalosporins. One hundred percent were resistant to sulfonamides and bacitracin zinc. The integrons were further confirmed by sequencing. No class II integrons were detected, whereas 50% (n = 16) of the A. pyogenes isolates were positive for the presence of the intI I gene, but only 13 contained gene cassettes. Sequence analysis of gene cassettes revealed 6 gene cassettes, 4 of which encode resistant determinants of aminoglycosides (aadA1, aadA5, aadA24, and aadB) and 1 of which encodes the resistance gene of chloramphenicol (cmlA6). The function of the sixth identified cassette, designated ORF1, is unknown. The gene cassette arrays aadA24-ORF1, aadA5, and aadA1-addB-cmlA6 were found in 46.13% (6/13), 38.46% (5/13), and 38.46% (5/13) of the isolates, respectively. These cassettes segregated according to a consistent pattern, with aadA5 always alone, ORF1 always with aadA24, and aadA1-aadB and cmlA6 always together. Most of the positive integrons existed in the multiresistant isolates (n = ∼3 to 7), indicating that the integrons played an important role in the dissemination and spread of antimicrobial resistance. This is the first report of A. pyogenes infections in dairy cows in China and of detection of gene cassettes and integrons in A. pyogenes.

BackgroundThe mcr-1 gene confers transferable colistin resistance. mcr-1-positive Enterobacteriaceae (MCRPE) have attracted substantial medical, media, and political attention; however, so far studies have not addressed their clinical impact. Herein, we report the prevalence of MCRPE in human infections and carriage, clinical associations of mcr-1-positive Escherichia coli (MCRPEC) infection, and risk factors for MCRPEC carriage.MethodsWe undertook this study at two hospitals in Zhejiang and Guangdong, China. We did a retrospective cross-sectional assessment of prevalence of MCRPE infection from isolates of Gram-negative bacteria collected at the hospitals from 2007 to 2015 (prevalence study). We did a retrospective case-control study of risk factors for infection and mortality after infection, using all MCRPEC from infection isolates and a random sample of mcr-1-negative E coli infections from the retrospective collection between 2012 and 2015 (infection study). We also did a prospective case-control study to assess risk factors for carriage of MCRPEC in rectal swabs from inpatients with MCRPEC and mcr-1 negative at the hospitals and collected between May and December, 2015, compared with mcr-1-negative isolates from rectal swabs of inpatients (colonisation study). Strains were analysed for antibiotic resistance, plasmid typing, and transfer analysis, and strain relatedness.FindingsWe identified 21 621 non-duplicate isolates of Enterobacteriaceae, Acinetobacter spp, and Pseudomonas aeruginosa from 18 698 inpatients and 2923 healthy volunteers. Of 17 498 isolates associated with infection, mcr-1 was detected in 76 (1%) of 5332 E coli isolates, 13 (<1%) of 348 Klebsiella pneumoniae, one (<1%) of 890 Enterobacter cloacae, and one (1%) of 162 Enterobacter aerogenes. For the infection study, we included 76 mcr-1-positive clinical E coli isolates and 508 mcr-1-negative isolates. Overall, MCRPEC infection was associated with male sex (209 [41%] vs 47 [63%], adjusted p=0·011), immunosuppression (30 [6%] vs 11 [15%], adjusted p=0·011), and antibiotic use, particularly carbapenems (45 [9%] vs 18 [24%], adjusted p=0·002) and fluoroquinolones (95 [19%] vs 23 [30%], adjusted p=0·017), before hospital admission. For the colonisation study, we screened 2923 rectal swabs from healthy volunteers, of which 19 were MCRPEC, and 1200 rectal swabs from patients, of which 35 were MCRPEC. Antibiotic use before hospital admission (p<0·0001) was associated with MCRPEC carriage in 35 patients compared with 378 patients with mcr-1-negative E coli colonisation, whereas living next to a farm was associated with mcr-1-negative E coli colonisation (p=0·03, univariate test). mcr-1 could be transferred between bacteria at high frequencies (10−1 to 10−3), and plasmid types and MCRPEC multi-locus sequence types (MLSTs) were more variable in Guangdong than in Zhejiang and included the human pathogen ST131. MCRPEC also included 17 unreported ST clades.InterpretationIn 2017, colistin will be formally banned from animal feeds in China and switched to human therapy. Infection with MRCPEC is associated with sex, immunosuppression, and previous antibiotic exposure, while colonisation is also associated with antibiotic exposure. MLST and plasmid analysis shows that MCRPEC are diversely spread throughout China and pervasive in Chinese communities.FundingNational Key Basic Research Program of China, National Natural Science Foundation of China/Zhejiang, National Key Research and Development Program, and MRC, UK.

To assess the prevalence of antimicrobial resistance and class I integrons in Escherichia coli strains (n = 58) isolated from bovine mastitis in Inner Mongolia, antimicrobial susceptibility and the presence of various types of integrons were characterized. Most isolates were susceptible to amikacin, colistin, ceftazidime, gentamicin and kanamycin, while those also exhibited high resistant incidence rates to ampicillin, amoxicillin, sulfadiazine and sulfamethoxydiazine. The integrase gene of integrons was amplified by PCR using degenerate primers. The integrons were confirmed by restriction fragment length polymorphism (RFLP) analysis of positive PCR products. Neither class II nor class III integron was detected, while 56.90% (n = 33) of the isolates were positive for the presence of intI1 gene. Sequencing analysis of gene cassettes revealed that seven gene cassettes were found, which encoded resistance to trimethoprim (dfrA1 and dfrA17), aminoglycosides (aacA4, aadA1 and aadA5) and chloramphenicol (catB3), respectively. Of them, the gene cassette array dfrA17–aadA5 was found most prevalent (62.96%). The percentage of positive-integron among the isolates whose resistant profile was relatively broad (n ≥ 7) is 100.00%, while the one in narrow-profile isolates (n = 2–6) is 30.56%. The correlation analysis revealed the incidence of integrons among the isolates were highly related to the resistant profile, indicating integrons play an important role in the dissemination and spread of the antimicrobial resistant strains.

The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr in Enterococcus species of swine origin. Twenty-five cfr-carrying Enterococcus isolates were collected from swine in Beijing, Guangzhou, and Shandong, China. The isolates consist of 24 Enterococcus casseliflavus and one Enterococcus gallinarum isolate, and exhibited six SmaI PFGE patterns. The cfr gene was located on plasmids in all isolates except E. casseliflavus En83, in which cfr was located on the chromosomal DNA. The cfr gene environments in most of these isolates contain DNA sequences similar to pEF-01, which was first found in Enterococcus. However, inverse PCR analysis suggested that the cfr-carrying circular forms might be different from pEF-01. The circular forms in Eg51 and its transconjugant, and En23, En10, and En94 are similar to the circular form in pEF-01, except for the truncated IS1216, which is replaced by a transposase of the IS256 family in En24. The cfr circular form could not be detected in either En77 or En83, and the same cfr-carrying segments of ∼10 kb had only 3500 bp of sequence similar to pEF-01. This is the first report of cfr gene in E. casseliflavus and E. gallinarum. The potential dissemination of the multidrug resistance gene amongst different bacterial species, especially in enterococci of human and animal origins, is concerning and should be closely monitored.

The present study aimed to determine the prevalence and mechanisms of macrolide–lincosamide (ML) resistance in 72 Staphylococcus aureus isolates from cows with clinical mastitis. Minimum inhibitory concentrations (MIC) of ML antibiotics were determined by the broth microdilution technique, inducible ML resistance phenotype by the D test, and ML resistance genes by PCR assay. The isolates showed a high level of resistance to erythromycin (93.1%), azithromycin (93.1%), spiramycin (41.7%), tylosin (40.3%), tilmicosin (27.8%), and clindamycin (36.1%). Macrolide–lincosamide MIC90 values were ≥128 mg/L. Inducible ML resistance (iML) phenotype was detected in 52.8% (38/72) of isolates. In erythromycin-resistant (ER-R) strains, methylase genes ermB and ermC, efflux gene msrA/msrB, and inactivating enzyme genes lnuA and mphC were present alone or in various combinations, with ermB and ermC genes predominating. This is the first report of ML resistance genes ermB, mrsA/mrsB and mphC in S. aureus isolated from bovine mastitis. The occurrence of high levels of resistance to ML antibiotics among the S. aureus isolates, and the high rate of iML phenotype, indicate that appropriate alternative antibiotics should be prescribed for treating bovine mastitis caused by S. aureus. Furthermore, significant differences in the conformations of lactone rings of 16- and 14-membered macrolides could explain why some isolates with a constitutive ML resistance (cML) phenotype were sensitive to 16-membered macrolides alone. The different interaction of the 16-membered macrolides with the 50S ribosomal subunit is also presumably the reason why the susceptibility results of tilmcosin differed from those of tylosin and spiramycin.

•We characterised the Salmonella serovars in food-producing animals in Shandong, China.•Salmonella on poultry carcasses were from the cross-contamination in abattoir.•Antibiotic resistance rates in 2012 were significantly higher than those in 2009.•In 2012, 41.5% of Salmonella were resistant to ciprofloxacin.•First report of multidrug resistant Salmonella Indiana isolated from ducksThe aims of this study were to investigate the serotype distribution, genetic relationships and antibiotic resistance of Salmonella from food-producing animals in Shandong province of China in 2009 and 2012. A total of 362 out of 1825 samples from chickens, 53 out of 445 samples from ducks, and 50 out of 692 samples from pigs were positive for Salmonella. Isolates were subjected to serotyping, antibiotic susceptibility testing (15 antibiotics) and pulsed-field gel electrophoresis (PFGE). The most common serotypes recovered in the chicken samples were Enteritidis (n = 294, 81.2%) and Indiana (n = 45, 12.4%). For ducks, Cremieu (n = 25, 47.2%), Indiana (n = 13, 24.5%) and Typhimurium (n = 9, 17%) were frequently isolated. In the pig samples, Derby (n = 29, 58%), Typhimurium (n = 9, 18%), and Enteritidis (n = 6, 12%) were the most common serovars. PFGE results indicated that clonal dissemination of each serovar was prevalent, and that the Salmonella found on the poultry carcasses was caused by cross-contamination in the abattoirs. More than 99% of the Salmonella isolates collected were resistant to at least one antibiotic. The Salmonella resistance rates for 15 antibiotics in 2012 were significantly higher than those in 2009. In 2012, the highest resistance was to nalidixic acid (95.9%), followed by sulphafurazole (78.2%) and ampicillin (72.3%); the lowest levels of resistance were to kanamycin (40.1%) and amikacin (38.7%). Additionally, 41.5% and 42.2% of the Salmonella were resistant to ciprofloxacin and ceftiofur, respectively. Noticeably, 25% of the serovar Enteritidis and all of the serovar Indiana were resistant to at least 10 antibiotics in 2012. The increasing trend of antibiotic resistance in Shandong province indicates the need for more careful use of antibiotics.

To evaluate the distribution of most known staphylococcal superantigen (SAg) genes in Staphylococcus aureus isolated from bovine mastitis cases, a genetic analysis of 15 SAg genes and genotypes was performed in a total of 283 S. aureus isolates collected from milk samples of cows with subclinical mastitis in two major diary production regions of China. Almost 65% of the isolates possessed at least one toxin gene. The most frequently found genes were sea (36.0%) followed by sei (31.8%), seg (31.4%) and selm (26.9%). The genes see, selk, or selo were not found in any of the isolates tested. Overall, 28 SAg genotypes were observed, among which the genotypes sea–seg–sei–selm, seg–sei–selm–seln, and sea–sed–selj predominated at the rate of 8.8%, 7.4%, and 6.7%, respectively. Marked geographical variations were noticed in the distribution of individual SAg genes and genotypes among S. aureus isolates from the two different regions. The relationship between toxin genotypes and toxin genes encoding profiles of mobile genetic elements (MGEs) was analyzed, revealing that majority of SAg genes were present in certain MGEs, which were in accordance with current knowledge about MGEs carrying those genes. However, some gene combinations suggest the possibility of the existence of variants or new types of MGEs.

A competitive indirect chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for florfenicol (FF) and its major metabolite florfenicol amine (FFA) residues in chicken muscle has been developed and validated according to Commission Decision 2002/657/EC criteria. The IC50 value of the method was 0.153 μg kg−1 for FFA with a cross-reactivity of 74.3% for FF under optimum conditions, in which FFA–F–BSA (FFA–formaldehyde–BSA) and FF–G–OVA (FF–glutaric anhydride–OVA) were used as an immunogen and a coating antigen, respectively. FFA and FF were easily extracted from chicken muscle with a 40:1 ethyl acetate–ammonia mixture, obtaining recoveries of 70.3–100% (FFA) and 71.8–102.0% (FF). Accuracy, precision, selectivity, robustness, limit of detection (LOD), limit of quantification (LOQ) and detection capability (CCβ) of the assay have been assessed during the validation process. LOD values in chicken muscle were 0.353 μg kg−1 for FFA and 0.526 μg kg−1 for FF (10-fold dilution) and 0.453 μg kg−1 for FFA and 0.657 μg kg−1 for FF (100-fold dilution). Furthermore, the CL-ELISA method gave CCβ values of 1.0 μg kg−1 for FFA and FF. Finally, real chicken muscle samples were analyzed with the CL-ELISA method, traditional ELISA and a previously reported gas chromatography-negative chemical ionization mass spectrometry (GC-MS), and results confirmed the utility of this new CL-ELISA for trace determination of FF and FFA, simultaneously.

Chloramphenicol (CAP) is a forbidden antibiotic that enters the food chain by illegal use in food-producing animals, potentially causing aplastic anaemia in humans. Immuno-PCR has the potential to address the need of meeting strict limits for this antibiotic by detecting trace levels of CAP present in animal-derived foods. A real-time immuno-quantitative PCR (RT-IPCR) assay for the quantification of CAP based on the simple and quick immunomagnetic bead recovery of CAP in milk was developed. The immunomagnetic beads were obtained by linking the reporter DNA to anti-CAP monoclonal antibody with N-succinimidyl S-acetylthioacetate (SATA) and succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and immobilising the anti-CAP monoclonal antibody/DNA-label conjugate on the magnetic beads with 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (SNHS). The RT-IPCR assay allows the sensitive detection and quantification of CAP in the concentration range of 0.001–0.11 μg L−1 (R2 = 0.9986) with a 50% inhibition concentration (IC50) value of 0.008 μg L−1. The RT-IPCR approach discussed here is presented as a model system that could be easily adapted for small molecule detection in a variety of foods using simple immunomagnetic bead recovery.

Three immunizing haptens, 6-(4-aminophenylsulfonamido)hexanoic acid (HS), 4-(4-(4-aminophenylsulfonamido)phenylsulfonamido)benzoic acid (SSS) and 4-(4-(4-aminophenylsulfonamido)phenyl)butanoic acid (SA10), were synthesized and used as haptens to produce generic polyclonal antibodies against sulfonamides. A novel generic enzyme-linked immunosorbent immunoassay (ELISA) for the detection of sulfonamides was developed based on one site heterologous coating hapten derived from sulfadiazine (STZ), providing IC50 values below 100 ng mL−1 for the 26 sulfonamides tested in buffer. The study showed that the site heterologous coating hapten could significantly improve the sensitivity of ELISA in comparison to a homologous or bridge length heterologous coating hapten. The developed ELISA was used first for the detection of five sulfonamides in spiked chicken muscle with recovery values ranging from 80.9 to 87.4% when the clean-up step was not included and from 85.6% to 92.7% when the clean-up step was included in sample pretreatment. The study showed that the high sensitivity and broad specificity of the assay make it a suitable screening method for the determination of multi-sulfonamides residues in chicken muscle.