Monica Ermolli

Adelina Prospero

Branko Balla

Maddalena Querci

Alessandra Mazzeo

Guy Van Den

PROTEINS DETECTION AND QUANTIFICATION

Keywords: GMO, immunoassay, ELISA Reverse, Cry1AB, CP4EPSPS

An innovative immunoassay, called ELISA Reverse, based on a new conformation of the solid phase was developed. The solid support was expressly designed for being immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified (GM) maize was optimised. The method was tested using GMO concentrations from 0.1 to 2.0%. The LOD and LOQ of the method were respectively determined equal to 0.0056 and 0.0168 (expressed as % GMO content). A qualitative multiplex assay to assess the presence of two GM proteins simultaneously has also been established for the case of the Cry1AB and the CP4EPSPS present in GM maize and soy respectively.

INTRODUCTION

Development and optimisation of methods and protocols for detecting and quantifying genetically modified organisms (GMO) in food and feed is one of the main activities of the European Commission Biotechnology & GMOs Unit. The work reported here takes into consideration the current need for monitoring the presence of several contaminants in food and feed products. In compliance with EU requirements for assuring food safety and traceability, several tests have to be performed. In particular for GMOs Regulation (EC) 1830/2003 concerns the traceability and labelling of products containing traces of GMOs at or above the fixed 0.9% legal threshold. Following EU requirements concerning the traceability and labelling of products containing traces of GMOs, a protocol for the simultaneous detection of different GMO-related proteins was developed in the form of an innovative and convenient solid phase immunoassay.

ELISA Reverse (ER) is a new method and the device has been patented by the Science and Technological Park, Moliseinnovazione (Patent number PCT WO 03/085401 A1).

The ER was developed on the basis of an immunosorbent plastic device allowing the introduction of the solid phase of an ELISA reaction directly into the liquid sample and therefore solving several practical problems linked to the traditional ELISA format.

Briefly the ELISA Reverse solid phase (device) is constructed of polystyrene pins protruding from a rod and positioned at modular distance. Each pin can be used to detect a specific target therefore the number of targets detectable depends on the number of pins present on the device. Multiplex assay and single detection can be performed following the same protocol; the only difference being the secondary conjugated antibody chosen according to the aim of the assay. The pins can be immersed directly in samples collected in individual tubes of variable volume, or distributed into 96 wells micro-plates (devices at 4 ÷ 12 pins) or 384 wells micro-plates (devices at 8 ÷ 24 pins). As in the common ELISA procedure the protocol consists of one hour incubation time in the presence of the target followed by three washing steps. Then the incubation step in the presence of the secondary antibody is commonly 16-20 min followed by second cycle of washing and the colour development step of 10 min. All these steps of the ER procedure can be performed in micro-plates; the number of the micro-plates used during the whole procedure is defined each time according to the number of devices used for the assay (figure 1).

In a previous study the ELISA Reverse was successfully applied for the simultaneous detection of different antibodies in bulk milk tested for the presence of Salmonella, bovine rhinotracheitis (IBR) and bovine leucosis (EBL) infections [START_REF] Mazzeo | ELISA Reverse Technology per il controllo di paramentri sanitari negli allevamenti da latte[END_REF].

The method was found to be reliable and versatile for the simultaneous detection of multiple targets allowing the possibility to increase the sample volume enhancing method sensibility, reducing costs, simplifying samples handling, and the washing/drying steps of the solid phase.

The technology offers additional advantages: avoiding sample concentration, no specific lab environment is required; coupled dried devices can be stored as the ELISA plate at 4ºC for several months, the protocol for multiplex detection is handily and fast as the single target detection. Quantitative analyses can be performed using a microplate reader as for the ELISA assays As for all immunoassays, the limiting factor is the dependence on the availability of suitable antibodies or proteins for the sensibilization of the pins. In this publication, the aim was to explore the possibility to detect traces of the Cry1AB protein contained in some GM Maize lines (e.g. Bt11 and Mon810) and to measure the quantity the GM protein within the range of 0.1 to 2.0% (weight GMO/weight). Secondly, the ability to use the device for multiplex assay was evaluated after binding two different GM specific antibodies on the same device i.e the Cry1AB and CP4EPSPS as examples of the most representative GM proteins present on the EU food market.

MATERIALS AND METHODS

Antibodies: specific monoclonal antibodies against Cry1AB were kindly provided by Monsanto (USA). Mouse IgG monoclonal antibodies against CP4EPSPS were produced by Moravian Biotechnology (Czech Republic). HPRT labelled monoclonal secondary antibodies anti selected GM proteins were purchased from Strategic Diagnostic (USA) and used as described by the product's guide (cat. n. 7110000 and 7100000).

Samples: Bt11 Maize Standard material at 0%, 0.15%, 0.5% and 2% were purchased from Strategic Diagnostics Inc. (USA) (cat. n. 7110000). Serial dilutions of the 2% standard in PBS buffer were prepared to obtain the 0.1, 0.2, 0.3, 0.4, and 0.5% Bt11 concentrations also tested in the experiments. Certified Reference Materials (CRMs) for genetically modified Roundup Ready soya beans (ERM-BF410) at 0% and 1%, as well as Certified Reference Materials (CRMs) for genetically modified Bt-11 maize (ERM-BF412) at 0% and 1% were from IRMM (Geel, Belgium).

Plastic support: prototypes of polystyrene activated devices HB10 were produced by Biomat snc (Rovereto, Italy) while low binding micro-plates were purchased from Thermo Electron Corporation (Finland).

Other reagents: 3,3',5,5' tetramethylbenzidine (TMB) liquid substrate system for ELISA (cat. n. T0440) was purchased by Sigma Aldrich (Italy). Lysis buffer was 10 mM Sodium Borate buffer pH 7.5.

Assay conditions. CP4EPSPS device coating step: The optimal amount of capture antibodies to be used was estimated by testing 25, 50, and 100µg/mL of total proteins of the hybrydomas surnatant containing the monoclonal antibodies against CP4EPSPS. 100µg/mL was the most reliable amount of total proteins (see Results and Discussion session and table 1). Pins sensibilization was performed over night at 4°C by dispensing in the low binding micro-plates 100µL of carbonate buffer pH 9.6 containing 100µg/mL of total proteins. Cry1AB device coating step: 100µL of carbonate buffer containing 10µg/mL of purified antibodies against Cry1AB were used. The blocking step was performed in a micro-plate in presence of PBS buffer containing 5% non-fat dried milk for two hours at room temperature (Crowther, 2000). The coupled dried devices can be stored at 4ºC up to three months. Samples: Preparation of samples for the Cry1AB quantitative assay was performed by homogenizing 1g of each maize standard (0%, 0.15%, 0.5% and 2%) in 4 mL of lysis buffer. Samples containing 0.1, 0.2, 0.3, 0.4, and 0.5% GM Maize were obtained by serial dilutions of the 2% standard in PBS buffer.

Samples used for the Cry1AB and CP4EPSPS qualitative assays consisted in maize and soy at 0% and 1% GM content. Maize samples were extracted as previously described; while soy samples were prepared by homogenizing 0.45 g of each standard in 4 mL of lysis buffer. Samples were then centrifuged at 5300 x g for 15 min.

Quantitative and qualitative assay protocol: Following lysis, 100µL sample surnatant were transferred in triplicate into the micro-plate wells and pins were introduced and incubated for one hour at room temperature. The devices were then removed and washed at least three times before being immersed into 100µL of the secondary-conjugated antibody solution (dilution 1:10000) previously distributed in a clean micro-plate.

Incubation step was at room temperature for one hour. After incubation and three rounds of washing, the devices were incubated in 100µL of TMB substrate for 20min. for the visualization of the complex. 100µL of stopping solution (1N Sulfuric acid) were then added to each well to block the reaction. Absorbance values were immediately read by Bio-Rad micro-plate reader at 450 nm.

Data analysis was performed according to the aim of the test. In all experiments one pin of the device was used as "blank".

All experiments were repeated at least three independent times following the same experimental design.

RESULTS AND DISCUSSION

We have successfully coupled both monoclonal antibodies at our disposal to the solid supports following the protocol previously described. The devices at our disposal were designed with four pins as shown in figure 1. All tests reported in the present document were conducted three times and all samples were measured in triplicate. For the development of the quantitative assay the solid supports were coupled with specific monoclonal antibodies recognizing the Cry1AB protein. The test was optimized to match standard requirements imposed for GMO analysis (EN ISO 21572:2002[START_REF] Paoletti | Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing[END_REF]. In particular, the test conditions were optimized in order to reach values of LOD and LOQ allowing the detection and quantification of contamination around the 0.1% level as the conventional ELISA tests (Ermolli et al., 2005).

Cry1AB quantitative assay was performed within the range of 0.1 to 2% of GM maize content as shown in figure 2 and table 1.

[Insert figure 2 and table 1 about here]

To fine tuning the method performance and to verify its ability to distinguish among samples with close GMO content values at low concentrations, the assay was also performed with samples at 0.1, 0.2, 0.3, 0.4 and 0.5% (figure 3 andtable 2).

[Insert figure 3 and table 2 about here]

In both cases the parameters considered for the evaluation of the method were accuracy, repeatability, SD, RSD, LOD and LOQ according to the level of GMO content (tables 3 and 4). Our results indicate a very good performance of assay: the linearity achieved within the range of 0% to 0.5% was confirmed when the same analysis was performed from 0% to 2% as reported in figures 2 and 3 and tables 2 and 3. The limits of detection (LOD) and of quantification (LOQ) were expressed in % of GMO content and calculated as follows: LOD = [ b x (Blank) + 3x SD b (Blank)]/q 1 (q 1= slope of the regression line), LOQ = [ b x (Blank) + 9x SD b (Blank)]/ q 1 . Considering the slope of the regression line calculated from 0.1 to 0.5%, LOD was equal to 0.0054 and LOQ value was 0.0162. In the same way the limits of detection and quantification calculated considering the slope of the regression line from 0.1 to 2% showed comparable values, 0.0056 and 0.0168 respectively. Method accuracy is defined as the closeness of agreement between a test result and the accepted reference value. Method accuracy was calculated using the formula (Observed Value / Theoretical Value) x 100 and expressed in %. Its value is considered acceptable within ± 25% of the accepted theoretical value. As shown in tables 3 and 4, method accuracy was within the accepted range, achieving values from 86.52 to 113.47. The repeatability standard deviation (RSD) expressed by the formula RSD r = SD r / x (in %) was used to characterize the assay performance under repeatability conditions. The values obtained in our experiments were significantly lower than the acceptable threshold of 25% [START_REF] Paoletti | Definition of Minimum Performance Requirements for Analytical Methods of GMO Testing[END_REF].

[Insert tables 3, 4 about here]

A qualitative multiple protocol to assess the presence of two GM proteins simultaneously has also been established for the Cry1AB and the CP4EPSPS proteins. In this case, two of the four pins of the device were coupled with the antibody recognising EPSPS and the other two pins with the antibody specific for Cry1AB.

The pins of the devices were coupled as indicated in the Materials and Methods session.

For the CP4EPSPS a preliminary titration step was required to establish the optimal coupling conditions. Three different amounts of total proteins of the hybrydomas medium were tested and the results analysed by parameters, from the infectious diseases and contaminants to the GMO detection and quantification, need to be taken in to consideration before the commercialisation of a food product and consequently various analyses have to be performed.

The threshold limits imposed to an assay for food analysis have been thoroughly evaluated in the conduction of our study and our results are in line with the expectations confirming the potentiality of the method. The present document reports the application of the device and of the relative assay for GMO analysis; the results obtained, the versatility and flexibility of the method justify further development of the method with the inclusion of other significant food safety targets. 3. The device is removed and washed (three to five passages in wells containing washing solution). 4. The pins are then immersed into the wells pre-filled with specific conjugate(s) antibodies and incubated for 20min. 5. The device has to be washed as in step three. 6. The pins are immersed in wells pre-filled with chromogenous substrate and incubate at room temperature for 10min. 7. After the colour development step the device is removed from the wells and the plate can be immediately analysed by a micro-plate reader. ss -significant at the 0.05 level s -significant at the 0.1 level ns -not significant at the 0.1 level Table 5. We titrated the optimal amount of capture antibodies to be coupled to the devices testing 25, 50, 100µg/mL of total proteins of the hybrydomas surnatant containing monoclonal antibodies against CP4EPSPS. The results for three different pairwise tests used by ANOVA acquired by SAS software proving the significant difference between 0% GMO level and other GMO tested levels. 100µg/mL was the selected amount. * samples measured on the following levels with GMO content (in %) -0, 0.1, 0.2, 0.3, 0.4 and 0.5 ** samples measured on the following levels with GMO content (in %) -0, 0.3, 1.25 and 2.5 ss -significant at the 0.05 level s -significant at the 0.1 level Table 6. Qualitative multiplex assay: the results of three different pair-wise tests used by ANOVA acquired by SAS software proving the significant difference between 0% GMO level and 1% GMO level in samples containing a mixture of GM soy and GM maize.

F

Figure 1 .

 1 Figure 1. ELISA Reverse technology. For experimental trials, prototypes of immunosorbent devices were realized in polystyrene HB10, having conformation of rods

  . A) Single device displayed close to a coin of one euro to appreciate the device shape and dimensions. B) Devices positioned inside the low binding micro-plate during the colour development step. C) Scheme of the ER procedure.

Figure 2 .Figure 3 .

 23 Figure 2. Weighted ordinary least squares regression for Cry1AB quantitative test.Association between optical density and % of GMO content obtained for the range from 0% to 2% of Cry1AB.

F

  3 different pair-wise tests (t-test, Tukey's test, Bonferroni t-test) in ANOVA acquired in SAS software. As reported in table 1, the tests gave significant difference between the blanks and the GM samples only if 100µg/mL of total proteins were used.both at 1% GMO level. Results were statistically evaluated to verify the ability of the assay to distinguish between 0% to all others measurements performed at 1% GMO level of contamination. The assay was reliable and the results of three different pair-wise tests

	(t-test, Tukey's test, Bonferroni t-test) used by ANOVA acquired by SAS software
	confirmed the data.
	[Insert table 6 about here] F o When applicable, protein-based methods have the potential to serve as cost-efficient,
	r practical and quick tools for screening and for multiplex analysis especially for routine
	P controls on large amount of samples.
	e e r
	R e v i e w
	O n
	l y

[Insert table 5 about here]

The results of the multiple screening tests are summarised in table 5. The assay was tested on 0% samples and on samples containing a mixture of GM soy and GM maize, In particular, multiple screening facilitates and speeds up the full characterization of food products and of the single ingredients, as well as the identification of crosscontamination. To comply with the different requirements established by current EU legal frame on food control and safety (Council Regulation (EC) No 1139/98, Directive 2003/99/EC, Directive 2004/43/EC, Commission Regulation (EC) No 466/2001) several

Table 1 .

 1 Cry1AB quantitative test: summary from the weighted ordinary least squares regression: regression coefficients, standard errors, p-values and confidence levels for 95% probability in the range from 0% to 2% GMO content.

		Weighted ordinary least squares regression *
		Coefficient	Standard Error	95% probability p-value Lower limit Upper limit
	Intercept	0.1484	0.0186	< 0.0001	0.1070	0.1898
	% of GMO content	1.5504	0.1029	< 0.0001	1.3210	1.7797
	* acquired by SAS software
	o
	r
	P
	% of GMO content	e Ordinary least squares regression * e r w e i v e Coefficient Standard Error p-value Lower limit Upper limit 95% probability R 1.7084 0.0464 < 0.0001 1.6089 1.8078
				O n
				l y

Table 2 .

 2 Cry1AB quantitative test: summary from the ordinary least squares regression for Cry1AB quantitative test: regression coefficient, standard error, p-values and confidence levels for 95% probability in the range from 0% to 0.5% GMO content.

	Level	% of GMO content Theoretical value Observed value	Accuracy (%)	Mean of OD	Repeatability characteristics SD RSD (%)
	1	0			0.0992	0.0111	11.18
	2	0.15	0.1702	113.47	0.4123	0.0085	2.05
	3	0.5	0.4929	98.58	0.9125	0.0448	4.91
	4	2	1.7304	86.52	2.8312	0.1154	4.08

Table 3 .

 3 Data represent the repeatability and accuracy of the results for each percentage level of GMO tested samples obtained by the Cry1AB quantitative test.

	Level	% of GMO content Theoretical value Observed value	Accuracy (%)	Mean of OD	Repeatibility characteristics SD RSD (%)
	2	0.1	0.1155	115.45	0.1973	0.0089	4.51
	3	0.2	0.2231	111.54	0.3811	0.0156	4.10
	4 5 6	0.3 F 0.4 o 0.5	0.3075 0.4235 0.4644	102.49 105.87 92.89	0.5253 0.7234 0.7934	0.0744 0.0561 0.0652	14.17 7.76 8.22
		r
		P
			e e r
			R e v i e w
					O n
						l y

Table 4 .

 4 Cry1AB quantitative test: repeatability and accuracy of the results for each percentage level of GMO tested samples.

		Multiple comparison tests
	Total proteins	t-test	Tukey's test	Bonferroni t-test
	25 µg/mL	S	s	ns
	50 µg/mL	S	s	ns
	100 µg/mL	Ss	ss	s

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ACKNOWLEDGEMENTS

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Amaia ; González-Salgado

González-Jaén Mª

Vázquez Covadonga

Belén Patiño

Ucm

Mª Teresa

González-Jaén;

Highly sensitive PCR-based detection method specific to Aspergillus flavus in wheat flour

Keywords: Aflatoxins, Mycotoxins -aflatoxins Aspergillus flavus, aflatoxin, polymerase chain reaction (PCR), detection, wheat, flour, internal transcribed spacer (ITS)

Introduction

Aspergillus Section Flavi contains a number of species capable of producing a wide array of toxins among which aflatoxins are the most important in food safety.

Aflatoxins are potent carcinogenic, mutagenic, and teratogenic secondary metabolites and are produced predominantly by Aspergillus flavus and Aspergillus parasiticus [START_REF] Bennett | The aflatoxigenic Aspergillus spp[END_REF]. This Section consists of two groups of species; one includes Aspergillus flavus, A. parasiticus, A. nomius and the recently described species A. pseudotamarii and A. bombycis (Cary and Ehrich 2006;[START_REF] Cary | Aflatoxigenicity in Aspergillus: molecular genetics, phylogenetic relationships and evolutionary implications[END_REF]), all of which produce aflatoxin. The other group includes the aflatoxin non-producing species A. oryzae, A. sojae, and A. tamarii, which have been used for production of traditional fermented foods in Asia [START_REF] Kumeda | Heteroduplex panel analysis, a novel method for genetic identification of Aspergillus Section Flavi strains[END_REF].

Aflatoxinogenic fungi can contaminate several food commodities including cereals [START_REF] Pittet | Natural occurrence of mycotoxins in foods and feeds-an update review[END_REF], peanuts [START_REF] Jelinek | Worldwide occurrence of mycotoxins in foods and feeds-an update[END_REF], spices [START_REF] Bartine | Growth and toxigenesis of Aspergillus flavus isolates on selected spices[END_REF] and figs [START_REF] Doster | Aspergillus species and mycotoxins in figs from California orchards[END_REF][START_REF] Färber | Detection of aflatoxinogenic fungi in figs by a PCR reaction[END_REF]. Foods and feeds are especially susceptible of colonization by aflatoxigenic Aspergillus species in warm climates where they may produce aflatoxins along several stages of the food chain: either at pre-harvest, processing, transportation or storage [START_REF] Ellis | Aflatoxin in food: occurrence, biosynthesis, effects on organisms, detection, and methods of control[END_REF]. The level of mold infestation and the identification of governing species are important indicators of the quality of the raw material and predict the potential risk for the presence of mycotoxins [START_REF] Shapira | Detection of aflatoxinogenic molds in grains by PCR[END_REF].

The traditional methods for identification and detection of these fungi in foods include culture in different media and morphological studies. This approach, however, is very [START_REF] Edwards | PCR-based detection and quantification of mycotoxigenic fungi[END_REF]. Moreover, these methods have low degree of sensitivity and do not allow the specification of mycotoxigenic species [START_REF] Zhao | Identification of Aspergillus fumigatus and related species by nested PCR targeting Ribosomal DNA internal transcribed spacer regions[END_REF]. PCR-based methods that target DNA are considered a good alternative for rapid diagnosis because of their high specificity and sensitivity, and have been used for the detection of aflatoxigenic strains of A. flavus and A. parasiticus [START_REF] Shapira | Detection of aflatoxinogenic molds in grains by PCR[END_REF][START_REF] Färber | Detection of aflatoxinogenic fungi in figs by a PCR reaction[END_REF][START_REF] Sweeney | The use of reverse transcription polymerase chain reaction (RT-PCR) for monitoring aflatoxin production in Aspergillus parasiticus 439[END_REF][START_REF] Criseo | Differentiation of aflatoxin producing and non-producing strains of Aspergillus flavus group[END_REF][START_REF] Chen | Polymerase chain reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A. parasiticus in peanut kernels by multiplex plymerase chain reaction[END_REF][START_REF] Mayer | Monitoring the production of aflatoxin B1 in wheat by measuring the concentration of nor-1 mRNA[END_REF][START_REF] Zachová | Detection of aflatoxigenic fungi in feeds using the PCR method[END_REF][START_REF] Somashekar | PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize[END_REF]). However, none of these methods has yet been able to reliably differentiate A. flavus from other species of the A. flavus group. In particular, A. flavus and A. parasiticus have two different toxicogenic profiles. A. flavus produces aflatoxin B1 (M1) B2, cyclopiazonic acid, aflatrem, 3-nitropropionic acid, sterigmatocystin, versicolorin A and aspertoxin, whereas A. parasiticus produced aflatoxin B1 (M1), B2, G1, G2 and versicolorin A [START_REF] Wilson | Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns[END_REF]. Another important fact is that Aspergillus flavus and A. fumigatus are responsible for 90 % of the aspergillosis in human beings [START_REF] Paya | Fungal infection in solid organ transplantation[END_REF].

Most of the PCR protocols are based on aflatoxin biosynthetic genes which are single copy and, therefore, less sensitive than multi-copy genes [START_REF] Bluhm | Multiplex Polymerase Chain Reaction assay for the differential detection of Trichothecene-and Fumonisinproducing species of Fusarium in cornmeal[END_REF][START_REF] Jurado | PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize[END_REF]). The intergenic spacer (IGS) and the internal transcribed region (ITS) from the rDNA units are multicopy (up to 100 or 300 copies per haploid fungal genome) and highly variable sequences which are widely used for phylogenetic studies and diagnostics of closely related fungal species such as Aspergillus [START_REF] Henry | Identification of Aspergillus species using internal transcribed spacer regions 1 and 2[END_REF][START_REF] Parenicová | Combined molecular and biochemical approach identifies Aspergillus japonicus and Aspergillus aculeatus as two species[END_REF][START_REF] Zhao | Identification of Aspergillus fumigatus and related species by nested PCR targeting Ribosomal DNA internal transcribed spacer regions[END_REF][START_REF] Varga | Molecular diversity of agricultural important Aspergillus species[END_REF][START_REF] González-Salgado | Discrimination of Aspergillus niger and other Aspergillus species belonging to section Nigri by PCR assays[END_REF][START_REF] Patiño | PCR detection assays for the ochratoxin-producing Aspergillus carbonarius and Aspergillus ochraceus species[END_REF] or Fusarium [START_REF] González-Jaén | Genetic markers for the analysis of variability and for production of specific diagnostic sequences in fumonisin-producing strains of F. verticillioides[END_REF]Mirete et al. 2004;[START_REF] Patiño | PCR detection assay of fumonisin-producing Fusarium verticillioides strains[END_REF]. In this study, we have developed a PCR protocol based on multi-copy sequences (ITS) specific to A. flavus which allows distinction from other aflatoxinogenic molds, in particular from A. parasiticus, and organisms commonly found on grains. The method proved to be very efficient even at short incubation times and at low spores concentration of A. flavus in wheat flour samples.

Materials and methods

Fungal isolates and culture conditions

A detailed description of the fungal strains used in this study is given in Table I. All fungal cultures were maintained on potato dextrose-agar (PDA, Scharlau Chemie, Barcelona, Spain) at 4ºC and stored as spore suspension in 15% glycerol at -80ºC. The isolates were cultured in 100 mL Erlenmeyer flasks containing 20 mL liquid medium Sabouraud (Scharlau Chemie, Barcelona, Spain). Cultures were inoculated with mycelial disks cut from the plates and incubated at 25ºC, 150 rpm. Mycelia from 2-dayold cultures were harvested by filtration through Whatman paper nº 1 and kept at -80ºC for DNA isolation.

[Insert Table I]

DNA extraction and PCR amplification

Genomic DNA of the strains was obtained using either the genomic DNA Extraction Kit (Genomix, Talent, Trieste, Italy), according to the manufacturer's instructions. All genomic DNAs used in this work were tested for suitability for PCR amplification using primers ITS1 and ITS4 [START_REF] Henry | Identification of Aspergillus species using internal transcribed spacer regions 1 and 2[END_REF], which amplify the ITS region in Aspergillus. The PCR reaction was performed in an Eppendorf Mastercycler Gradient

Detection of fungal spores in wheat flour

Molds are found in dry stored food and grains mostly as dormant structures, e.g., spores.

To test the ability of the PCR to detect A. flavus in food, different lots of commercially available ground wheat flour (1g each) were inoculated separately with 10 2 or 10 6 spores per g of A. flavus ITEM 4591. The flour was then resuspended in 5 ml of Sabouraud (Scharlau Chemie, Barcelona, Spain) with 1ml/l chloramphenicol and incubated in 10 ml sterile Falcon tubes on an orbital shaker at 28ºC and 150 rpm. The samples were incubated for 8, 16, 24 and 48 h respectively. Non-inoculated flour samples were used as negative control. After incubation, the tubes were subsequently centrifuged (5 min at 5000 rpm), the supernatant discarded and the pellets were frozen in liquid nitrogen. Total DNA was extracted and PCR reactions were carried out using the same protocols described above for pure cultures. All the experiments were made in triplicate.

Results

PCR specificity with FLA1/FLA2

Partial sequences of the ITS1-5.8S-ITS2 regions of A. flavus and other related Aspergillus species obtained in this and previous works and those retrieved from Data Bases (more than 25) were aligned. The design of a PCR assay is limited by the conserved regions available in the alignments and the suitable size of the amplicon to be easily detected in conventional agarose gels using ladder markers. A pair of primers, I were tested for amplification using the primer pair FLA1 and FLA2. A single fragment of about 500 bp was only amplified when genomic DNA from A. flavus strains was used (Figure 1), but not from other Aspergilli, including Aspergillus section Flavi species (A. tamarii, A. nomius, A. bombycis and A. parasiticus) or other Aspergillus (such as A. ochraceus or Aspergillus section Nigri). No product was observed with genomic DNA from other genera. Nonspecific products were not observed in any case. Control amplifications of the genomic DNA with primers ITS1 and ITS4 were positive for all the strains analysed.

PCR studies with inoculated wheat flour

In order to test the functionality of the method under practical conditions, an enrichment procedure was developed. In this procedure, Sabouraud-Chloramphenicol broth was added to inoculate wheat flour which was then incubated. The primer pair generated for the amplification of A. flavus was tested on samples after 8, 16, 24 and 48 h of incubation. Two levels of inoculated spores, 10 2 and 10 6 spores per g, were tested. Specific amplification was observed only for DNA extracted from wheat inoculated with A. flavus ITEM 4591 (Figure 2). A clear amplification product was observed when wheat flour was inoculated with 10 2 or 10 6 spores per g after 16, 24 and 48 h of incubation. No amplification products were obtained when DNA was extracted from non-incubated samples or incubated for 8h. The triplicates of the experiments showed no significative variation in the results.

Discussion

In this work, we have developed a PCR protocol specific for the detection of A. flavus to be used in a food matrix frequently contaminated with aflatoxins and other related Aspergillus species. This is the first protocol to our knowledge to specifically detect A. flavus by PCR amplification using a multycopy region (ITS). Discrimination of A. flavus from A. parasiticus is important because they produce different secondary metabolite profiles. The aflatoxins, cyclopiazonic acid, versicolorin and sterigmatocystin can all be produced by A. flavus while A. parasiticus is not known to produce cyclopiazonic acid and sterigmatocystin [START_REF] Wilson | Biology and ecology of mycotoxigenic Aspergillus species as related to economic and health concerns[END_REF]. Moreover, A. flavus was the prevalent species among Aspergillus section Flavi in most of the studies performed in several commodities [START_REF] Horn | Regional differences in production of aflatoxin b1 and cyclopiazonic acid by soil isolates of Apergillus flavus along a transect within the united states[END_REF][START_REF] Trucksessy | Occurrence of afatoxins and fumonisins in Incaparina from Guatemala[END_REF][START_REF] Vaamonde | Variability of aflatoxin and cyclopiazonic acid production by Aspergillus section flavi from different substrates in Argentina[END_REF][START_REF] Bankole | Natural occurrence of moulds and aflatoxin B1 in melon seeds from markets in Nigeria[END_REF]Melki Ben Fredj et al. 2006).

Although several PCR-based methods have been developed to detect aflatoxigenic fungi using aflatoxin biosynthetic genes they do not discriminate between A. parasiticus and A. flavus, and the amplification signal obtained is strong for A. parasiticus but it is weak for A. flavus [START_REF] Geisen | A multiplex polymerase chain reaction for the detection of potential aflatoxin and sterigmatocystin producing fungi[END_REF][START_REF] Shapira | Detection of aflatoxinogenic molds in grains by PCR[END_REF][START_REF] Zachová | Detection of aflatoxigenic fungi in feeds using the PCR method[END_REF]. This could be due to the similarity of the aflatoxin biosynthesis genes used as targets for specific primers in these two species [START_REF] Yu | Comparison of the omt A genes encoding O-methyltransferases involved in aflatoxin biosynthesis from Aspergillus parasiticus and A. flavus[END_REF][START_REF] Scherm | Detection of transcripts of the aflatoxin genes aflD, aflO and aflP by reverse transcription-polymerase chain reaction allows differentiation of aflatoxin-producing and non-producing isolates of Aspergillus flavus and Aspergillus parasiticus[END_REF]. The existing variability in ITS region we have used seemed to be sufficient to discriminate among these closely related species and, on the other hand, provided higher sensitivity than single copy genes. Indeed, the PCR assay developed for A. flavus did not cross react with A. parasiticus.

Moreover, robustness of the specific PCR assay for A. flavus was high according to the positive PCR amplification obtained with a wide range of template concentration (10pg-10ng) using either DNA from pure cultures or from contaminated sample. It has been estimated a sensitivity for multicopy sequences above 5.5 pg and for single copy sequences above 87.5 pg on agarose gel [START_REF] Jurado | PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize[END_REF]. A similar approach was The FLA1/FLA2 primers seemed to be more efficient to specifically amplify the A. flavus target sequence. These primers are located in a more variable region of the ITS.

Other approaches, such as PCR-RFLPs of ITS region, have been followed to discriminate both species, but are more laborious [START_REF] Somashekar | PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize[END_REF]).

However, this protocol does not allow the discrimination between A. flavus and A. oryaze. Molecular methods, including isoenzyme analyses and DNA/DNA hybridization studies, and DNA sequencing and comparison of the whole genome have confirmed that both species are almost genetically identical [START_REF] Kurtzman | DNA relatedness among wild and domesticated species in Aspergillus flavus group[END_REF][START_REF] Egel | Relationship among isolates of Aspergillus section Flavi that vari in aflatoxin production[END_REF][START_REF] Geiser | The phylogenetics mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae[END_REF][START_REF] Payne | Whole genome comparison of Aspergillus flavus and A. oryzae[END_REF][START_REF] Hedayati | Aspergillus flavus: human pathogen, allergen and mycotoxin producer[END_REF], with the exception of few differences in genes involved aflatoxin biosynthetic pathway [START_REF] Geiser | The phylogenetics mycotoxin and sclerotium production in Aspergillus flavus and Aspergillus oryzae[END_REF]. On the other hand, while A. flavus is a common environmental organism, A. oryzae is a 'domesticated' fungus and it would not be present in the niches where A. flavus occurs. Specific diagnosis in pure cultures is generally easier than directly on commodities where a complex mycobiota is present, often containing closely related species and several compounds which may affect the efficiency and sensitivity of the detection [START_REF] Rossen | Inhibition of PCR by components of food samples, microbial diagnostic assays and DNA-extraction solutions[END_REF][START_REF] Färber | Detection of aflatoxinogenic fungi in figs by a PCR reaction[END_REF] and in some cases additional incubation of the food samples for several days is required [START_REF] Chen | Polymerase chain reaction mediated characterization of molds belonging to the Aspergillus flavus group and detection of A. parasiticus in peanut kernels by multiplex plymerase chain reaction[END_REF]. Since molds are found mostly as asexual spores or dried mycelia on dry food, which contain only small amounts of DNA and are resistant to cellular disruption for DNA extraction (Shapira et an enrichment method to be used in wheat flour contaminated by spores. This integrated protocol allowed us to detect 10 2 spores per g after 16 h of incubation. This incubation time was shorter than the reported by other authors [START_REF] Shapira | Detection of aflatoxinogenic molds in grains by PCR[END_REF], and the detection limit high in our study. Although genomic DNA of wheat and contaminating fungi may become severely damaged during the manufacturing process, PCR assays could be particularly suitable to detect fungal contamination in flour because of the small size of the target genomic DNA. Previous reports describing PCR assays with amplicons of similar length indicate successful amplification using DNA isolated from flour (maize) [START_REF] Jurado | PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize[END_REF][START_REF] Somashekar | PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize[END_REF]. These evidences, and the high copy number of the target sequence, increase the probability of available template even after processing of wheat grains. On the other hand, very often contamination by post-harvest fungi occurs after processing of the seeds in the warehouse.

In conclusion, the PCR based protocol reported in this work is a rapid and powerful tool to detect A. flavus in certain food systems. The PCR assay and the sample preparation protocol were optimised to avoid the occurrence of false negative reactions.

  , laborious, and requires facilities and mycological expertise

  Hamburg, Germany) using 10 pg-10 ng of genomic DNA. The ITS1-5.8S-ITS2 sequences were obtained following the same protocol by[START_REF] Henry | Identification of Aspergillus species using internal transcribed spacer regions 1 and 2[END_REF] above mentioned and the PCR amplification products obtained and purify using the High Pure PCR Product purification Kit (Roche, Germany) according to the manufacturer's instructions. The PCR products were subsequently sequenced by both strands using the ABI PRISM DNA Sequencer (Applied Biosystems, Foster City, USA) according to the manufacturer's instructions in the Genomic Unit of the Complutense University of Madrid. These sequences and those retrieved from GenBank and other data bases were analysed and aligned by Clustal method using the program DNASTAR (Lasergene, Wisconsin, USA). Specific PCR assays were carried out using primers FLA1 (5' GTAGGGTTCCTAGCGAGCC 3') and FLA2 (5' GGAAAAAGATTGATTTGCG TTC 3') for A. flavus. PCR reactions were performed in an Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany). The PCR amplification protocol used for A. flavus was as follows: 1 cycle of 5 min at 95ºC, 26 cycles of 30s at 95ºC (denaturalization), 30s at 58ºC (annealing), 45 s at 72ºC (extension) and finally 1 cycle of 5 min at 72ºC. Amplification reactions were carried out in volumes of 25 µL containing 3 µL (10 pg-10 ng) of template DNA, 1.25 µL of each primer (20 µM), 2.5 µL of 10x PCR buffer, 1 µL of MgCl 2 (50 mM), 0.25 µL of dNTPs (100 mM) and 0.2 µL of Taq DNA polymerase (5 U/µL) supplied by the manufacturer (Biotools Madrid, Spain). PCR products were detected in 2% agarose ethidium bromide gels in TAE 1X buffer (Tris-and EDTA 1.0 mM). The 100 bp DNA ladder (MBI Fermentas, Vilnius, Lithuania) was used as molecular size marker.

  FLA1 and FLA2, specific to A. flavus was designed on the basis of the sequence the Aspergillus strains listed in Table

  et al. 2004 to identify A. flavus and another pathogenic species of the genus Aspergillus. They used ribosomal DNA and its flanking regions (including ITS1 region), but the efficiency of amplification in the case of A. flavus was still low.

  ), we have improved the sensitivity and applicability of our PCR protocol with

Figure 1 .

 1 Figure caption

Figure 2 .

 2 Figure 2. PCR-based detection of A. flavus using FLA1/FLA2 primers in wheat flour

  Fungal strains analysed indicating, origin, species, host and the occurrence of PCR amplification product with primers FLA1 and FLA2. Strains supplied by Dr. Sanchis (University of Lleida, Spain)(*). Strains supplied by Dr. Moretti (CNR, Bari, Italy)(**)

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Acknowledgments

This work was supported by the Spanish MCyT (AGL 2004-07549-C05-05/ALI) and by the project UCM-CM 910644. A. González-Salgado was supported by a Gobierno Vasco fellowship.


Martin Danaher

Katrina Campbell

Michael O'keeffe

Emiliana Capurro

Glenn Kennedy

Christopher T Elliott

Survey of the anticoccidial feed additive nicarbazin (as dinitrocarbanilide residues) 1 in poultry and eggs 2 3

Keywords: Chromatography -HPLC, Chromatography -LC/MS, Screeningbiosensor, Survey Additives/Contaminants: Residues Food Types: Animal products -meat, Eggs

A survey was carried out on the occurrence of dinitrocarbanilide (DNC) the marker residue for 16 nicarbazin, in poultry produced in Ireland during the years 2002 to 2004. Liver (n = 736) and breast 17 muscle samples (n = 342) were tested. DNC residues were found in 40% and 26% of liver and 18 breast muscle samples at levels greater than 12.5 and 5 µg kg -1 , respectively. DNC residues were 19 found at >200 µg kg -1 in 12 and 0% of liver and muscle samples, respectively. Samples of breast 20 muscle (n = 217) imported from 11 countries were also tested for DNC residues. A lower incidence 21 of DNC residues (6%) was found in imported breast muscle. Egg samples (n = 546) were tested 22 and DNC residues were found in nine samples, with levels ranging between 14 and 122 µg kg -1 .

23

Analysis of poultry, carried out as part of official food inspection in the period 2004 to 2006, 24 indicated a reduction in the number of broiler liver samples containing DNC at >200 µg kg -1 , to 25 approximately 7%. Low levels of DNC residues continue to be found in <2% of egg samples. 26 coccidiosis. Coccidiosis is an infectious disease caused by a microscopic protozoan parasite, 31 which damages the intestinal tract of the bird, causing illness and sometimes death. Intensively

Introduction

28

Nicarbazin is an equimolar mixture of 4,4΄-dinitrocarbanilide (DNC) and 2-hydroxy-4,6-29 dimethylpyrimidine (HDP). It is administered to poultry in feed for the prophylactic treatment of reared broilers are particularly susceptible to the disease, owing to the warm and humid conditions 1 of broiler houses. The disease is not as common when birds are raised under extensive conditions.

2

The licence for feed premixes containing nicarbazin as a single active ingredient was withdrawn 3 under Commission Regulation 2205/2001/EC [Anon. 2001]. Nicarbazin continues to be legally 4 marketed at levels of 40-50 mg kg -1 , together with the feed additive ionophore narasin, as the 5 combined product Maxiban® [Anon. 2007].

6

Nicarbazin is classified as a feed additive and not as a veterinary drug. No maximum 7 residue limits (MRLs) have been set for nicarbazin in the European Union (EU). Nicarbazin 8 depletion studies that have been carried out in broilers show that DNC is a more persistent residue 9 than HDP in edible tissues (Porter and Gilfillan, 1955). The FAO/WHO Joint Expert Committee on 10 Food Additives (JECFA) established an MRL of 200 µg kg -1 for DNC, as the marker residue, in 11 edible tissue (liver and meat) [Wells 1999]. The JECFA MRL for DNC has been adopted as the 12 action limit by regulatory authorities in the Republic of Ireland and in the UK. Liver samples are 13 tested for DNC residues in monitoring programmes on the island of Ireland as this tissue tends to 14 contain higher residue concentrations than muscle. No MRL has been defined for DNC in eggs 15 because nicarbazin is not approved for use in laying hens. An action level of 100 µg kg -1 DNC in 16 eggs has been adopted in the UK [Veterinary Residues Committee 2006].

17

The presence of DNC residues was first reported in poultry products nearly 20 years ago 18 [De Giovanni et al. 1989, Oishi andOda 1989]. Since then DNC residues have been reported in complex than eggs because nicarbazin is widely applied to broilers and several on-farm factors 1 that may contribute to residues. The contribution of on-farm factors to the DNC residues in broiler 2 tissues has been described in more detail elsewhere [O'Keeffe et al 2006a].

3

In the study reported here, DNC contamination of tissues and eggs has been investigated 4 in an integrated study across Ireland. Three surveys were organised to investigate the presence of 5 DNC in eggs, liver and muscle between the years 2002 and 2004. In addition, a study on DNC 6 residues in samples of imported poultry meat was undertaken.

3

Biosensor screening assays 4 Egg and liver samples were screened using a Biacore™ Biosensor assay [McCarney et al. 2003].

5

The assay was modified from the published assay to allow the sensitive detection of DNC residues 6 in muscle tissue.

8

Egg and liver assays 9

Homogenised egg or liver test samples (1 g) were weighed into 30 ml polypropylene tubes.

10

Acetonitrile (4 ml) was added to samples and they were vortexed (10 sec) and sonicated (2 min)

11

prior to centrifugation (1200×g, 10 min). The supernatants were transferred into glass test tubes 12 and evaporated to dryness under a stream of nitrogen at 60°C. Sample extracts were allowed to 13 cool for 1 min. At this point liver sample extracts were further purified. Liver extracts were and evaporated to dryness under nitrogen.

18

Egg or liver sample extracts were subsequently reconstituted by sequentially adding 19 methanol (200 µl) and HBS-EP buffer (800 µl), vortexing for 10 sec after each addition. Sample 20 extracts were transferred to eppendorf tubes and centrifuged (14,000×g, 5 min). Sample extracts 21 were further diluted 1:20 (v/v) in HBS-EP buffer prior to analysis on a Biacore TM Q system. Aliquots

21

Internal standard (20 µl of the 10 µg ml -1 d8-DNC working standard) was added to samples, which 22 were mixed and allowed to stand for 15 min prior to extraction. Samples were homogenised in 23 acetonitrile (8 ml) using a Silverson homogeniser for 40 sec and centrifuged (600×g, 10 min at 24 4°C). An aliquot of the supernatant (2.5 ml) was transferred to glass tubes (10 ml) and evaporated 25 to dryness under nitrogen at 60°C. After cooling, the residues were reconstituted in hexane (1 ml). phase, which consisted of 0.05M ammonium acetate in acetonitrile/water (75:25 v/v), was pumped 10 at a rate of 1.0 ml min -1 . The column effluent was split so that approximately 100 µl min -1 entered 11 the mass spectrometer. The run time for each injection was 7 min.

12 13 HPLC assays 14 Homogenised egg, liver or muscle samples (2 g) were weighed into 30 ml polypropylene tubes.

15

Fortified egg and liver samples were prepared at levels of 25 and 250 µg kg -1 by adding 50 µl 16 portions of 1 and 10 µg ml -1 DNC standard solutions to negative control samples, respectively. 17 Fortified muscle samples were prepared at levels of 10 and 100 µg kg -1 by adding 50 µl portions of 18 0.4 and 4 µg ml -1 DNC standard solutions to negative control samples, respectively. After 19 fortification, samples were allowed stand for 15 min prior to extraction. Acetonitrile (10 ml) were 20 added and samples were homogenised using a Polytron™. The homogeniser probe was washed 21 with acetonitrile (5 ml), which was retained. Samples were vortexed (2 min), sonicated (3 min) and 22 shaken (15 min), before centrifugation (1928×g, 10 min, 4°C). The supernatant was transferred to 23 a clean polypropylene tube and the sample was re-extracted as before using the acetonitrile (5 ml)

24

previously used to wash the homogeniser probe, plus water (1 ml). The supernatants were 25 combined and defatted using hexane (2 × 10 ml) by vortex mixing, centrifugation and removal of 26 the hexane layer. The acetonitrile layer was evaporated to dryness under nitrogen (60°C) and SPE cartridges (preconditioned with 2.5 ml acetonitrile and 2.5 ml acetonitrile/water (70:30, v/v)) 1 and eluted with 2.5 ml acetonitrile/water (70:30, v/v). The eluate from the cartridge was collected in 2 a glass test-tube. Egg and liver extracts were evaporated to dryness under nitrogen (60°C) and 3 reconstituted in acetonitrile/water (80:20, v/v, 500 µl). Muscle samples were reconstituted in 4 acetonitrile/water (80:20, v/v, 200 µl). Extracts were allowed to sit for 15 min prior to transfer to 5 HPLC vials; a phase separation may occur in some samples and care was taken not to transfer 6 this lower oily layer into the HPLC vials.

7

A 25 µl portion of the final sample extract was injected onto the HPLC system. The HPLC Samples from all surveys screened as containing DNC residues were confirmed by an 10 independent chemical assay (HPLC-UV or LC-MS/MS). HPLC-UV has been described as suitable 11 for confirming the presence of Group B substances, when used in conjunction with a second 12 independent detection assay (Anon. 2002). In this survey, an immunobiosensor was used as the 13 second independent detection assay.

15

Results and discussion

16

Liver survey 17

An overall summary of the poultry liver survey is presented in Figure 1. The survey showed that 12 18 and 3% of samples contained DNC residues at >200 and >1000 µg kg -1 , respectively. DNC 19 residues were found at <200 µg kg -1 in 28% of samples and no residues were detectable (i.e. not 20 detected above 33 µg kg -1 by immunobiosensor and/or confirmed above 12.5 µg kg -1 using 21 chemical assays) in 60% of samples.

23

Seasonal variation in levels of DNC in liver

24

The seasonal variation in DNC levels in broiler liver samples from eight companies (I to P) is 25 shown in Figure 2. In general, there was a similarity in the proportion of samples containing DNC 26 and of samples containing DNC at >200 µg kg -1 over time. In the period June to August the other months. A number of hypotheses may be proposed for the lower incidence of DNC residues 1 in poultry during the June to August period. One hypothesis is that reduced usage of nicarbazin 2 may have occurred during the summer months; nicarbazin can cause heat stress, resulting in 3 increased mortality in broilers during warm weather. A second hypothesis is that the lower 4 incidence of DNC residues may be related to the removal of licensing for products containing 5 nicarbazin alone that occurred in May 2002. A third hypothesis is that the higher incidence of DNC 6 residues in broiler liver from September onwards may have been due to changes in the feeding 7 programmes used on poultry farms, with nicarbazin-containing feed being given to birds closer to 8 slaughter time.

10

Variation in DNC levels in liver sampled from different companies 11

The breakdown by company of samples containing DNC residues and containing DNC residues 12 >200 µg kg -1 is shown in Table I. For two companies (F and G) no samples containing DNC 13 residues were found; however, few samples were sourced from these companies during the 14 survey. The percentages of samples containing DNC residues range between 15 and 84% for the 15 other companies. The mean value for 16 companies was 42% of samples contained DNC 16 residues. The mean result for companies containing DNC residues >200 µg kg -1 was 14%, with the lowest at 0% and the highest at 42%. Three companies, C, E and L had 10, 22 and 33% of 18 samples containing DNC residues but 0, 0 and 1% of samples from these companies contained show that DNC residues occur at higher concentrations in liver compared to muscle [Anon. 1999].

2

As a result, DNC residues may be detected for a longer period of time in liver and the probability of 3 finding DNC residues in tissue is lower for muscle than for liver. The results for the muscle and 4 liver surveys indicate that liver is the most appropriate matrix to use for monitoring DNC residues 5 because it is the matrix in which residues persist at highest concentrations. percentage of liver samples likely to contain DNC at >200 µg kg -1 was calculated (Figure 4); the 18 estimated incidence of liver samples containing >200 µg kg -1 DNC was similar to that observed for 19 the liver survey, with a mean value of 14% (Table I). I). Companies C and D showed no appreciable change in incidence between 3 the two surveys.

4

The proportion of liver samples containing DNC residues >200 µg kg -1 (determined for chemical assays) in 98% of egg samples. The levels of DNC residues present ranged between 14 1 and 122 µg kg -1 . DNC residues occurred in egg samples throughout the year. The contaminated 2 egg samples were sourced from four companies and one of these companies had five positive 3 samples. Nicarbazin is not approved for the treatment of layer hens and residues in eggs may be 4 attributed to contamination of layer feed with low levels of nicarbazin [Cannavan et al. 2000]. DNC 5 residues may be eliminated from eggs through adequate quality control of feed samples shipped to 6 farms.

8

Performance of the biosensor screening assay for detection of nicarbazin residues 9 Connolly et al. ( 2003) characterised the antibody applied in this assay. The antibody was shown to 10 be specific towards DNC and did not cross-react to other potential interfering substances namely, 11 the anti-coccidials, toltrazuril, halofuginone and ronidazole. In routine application, it has been found 12 that the antibody does not cross-react to lasalocid and other ionophore (monensin, salinomycin 13 and narasin) residues detected in egg and liver samples. The cut-offs for sending samples for 14 confirmatory analysis was determined through the analysis of 20 negative liver and 20 known 15 negative egg samples. Briefly, analysis of 20 known negative livers yielded values of 1.19 ± 5.32 16 µg kg -1 . The limits of detection (mean + 3 S.D.) and determination (mean + 6 S.D.) were calculated 17 as 17.1 and 33.2 µg kg -1 , respectively. A second validation study was carried out in eggs and 18 analysis of 20 known negative eggs yielded values of 3.07 ± 5.29 µg kg -1 . The limits of detection and determination were calculated as 18.9 and 34.8 µg kg -1 , respectively. A more comprehensive 20 evaluation of the suitability of the limits of determination was carried out during the egg and liver 21 surveys. A total of 145 samples confirmed to contain DNC residues by HPLC were also screened 22 by immunobiosensor. Evaluation of the results indicated that false negatives rates for the assay 23 are generally <5%. In the concentration ranges >33 -100 (n = 42 samples), >100 -200 (n = 62),

24

and >200 µg kg -1 (n = 41); 2, 1 and 0 false negative results were observed, respectively. This 25 corresponded to false negative rates of <5, <2 and 0%, respectively. It is highlighted that no false 26 negative results were observed at or around the MRL. Nicarbazin in liver (ug kg

  19 egg and liver samples tested in The Netherlands, UK, Italy and Ireland [Vertommen et al. 1989, 20 Veterinary Residues Committee 2006, Gallo and Serpe 1997, O'Keeffe et al. 2005]. A number of 21 studies have been carried out to identify the factors that may contribute to the presence of DNC 22 residues in eggs and edible tissues, such as feed contamination [Cannavan and Kennedy 2000,

[

  -dinitrocarbanilide (DNC) standard material was from Sigma-Aldrich (St. Louis, MO, USA). The 11 deuterium-labelled internal standard (d8-DNC) was custom synthesised by Quchem (Queen's 12 University, Belfast). Acetonitrile, methanol, dimethylformamide, water (all HiPerSolv grade), 13 dimethylsulphoxide and n-hexane (Analar grade) were obtained from BDH (Merck, Poole, Dorset, 14 UK). Cyclohexane (Pestican grade) was obtained from Labscan (Dublin, Ireland). DNC standard 15 stock solution (1 mg ml -1 ) was prepared in dimethylsulphoxide (Biacore and HPLC assays) and 16 DNC and d8-DNC standard stock solutions (1 mg ml -1 ) in dimethylacetamide (LC-MS/MS). The 17 biosensor and HPLC standard solutions were prepared every 3 months and were stored in amber 18 glass vials at room temperature in the dark preventing their solidification. LC-MS/MS standard 19 solutions were stored at 4°C and were stable for at least one month. The optical SPR Biosensor 20 system (Biacore TM Q) was obtained from Biacore TM (Uppsala, Sweden). Sensor chips (CM5), HBS-21 EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20) buffer and an 22 amine coupling kit were purchased from Biacore TM . Biacore control software, version 3.1, was used 23 for instrument operation and BIA evaluation software, version 3.0, for data handling. Chip surfaces 24 were prepared by immobilisation of a DNC mimic, glutamic acid (1-glutamic acid-(p-nitroanilide)), 25 which was obtained from Sigma (Poole, Dorset, UK). A DNC polyclonal antibody (R555) was used 26 for the biosensor assay. Production of the DNC antibody are described in detail elsewhere Connolly et al. 2002]. Bond Elut™ cartridges (C 18 , 500 mg, 3 ml) were from Varian (Harbor City, 1 CA, USA).

22

  of the extracts (50 µl) were mixed with an equal volume of the antibody and injected for 2 min over 23 the sensor chip surface at a flow rate of 25 µl min -1 . The response for the sample was determined 24 as the difference in the signal (Response Units, RU) measured before and after injection. The 25 surface was regenerated with a 1 min pulse of dimethylformamide/180 mM sodium hydroxide (5 ml). The supernatants were transferred into glass test tubes and 4 evaporated to dryness under a stream of nitrogen at 60°C. Sample extracts were allowed to cool 5 for 1 min. Muscle extracts were reconstituted by sequentially adding methanol (200 µl) and HBS-6EP buffer (400 µl) and vortexing for 10 sec after each addition. Reconstituted extracts were further 7 diluted (1:1, v/v) with HBS-EP buffer prior to analysis. In a deviation from the liver and egg assays, 8 three additional steps were required to regenerate the chip surface. It was found during the 9 development of the assay to detect low levels of DNC in muscle that there was noticeable carry 10 over of electrostatic nicarbazin residues between injections and a more intensive chip regeneration 11 protocol was required. Chip surfaces were allowed to contact sequentially with (a) 180 mM 12 NaOH/acetonitrile (80:20, v/v), (b) 100 mM HCl/acetonitrile (80:20, v/v) and (c) 180 mM 13 NaOH/acetonitrile (80:20, v/v) at a flow rate of 50 µl min -1 for 60 sec.

  DNC residues were confirmed by LC-MS/MS [Yakkundi et al. 2001] or by HPLC-17 UV [Capurro et al. 2005]. 18 19 LC-MS/MS assays 20 Homogenised liver samples (2.0 g) were weighed into 50 ml polypropylene centrifuge tubes.

26

  This was extracted by vortexing (10 sec) with methanol/water (75:25 v/v, 250 µl). The tubes were 27 centrifuged (600×g, 10 min at 4°C) and an aliquot (175 µl) of the aqueous layer was transferred to 28 . Calibration curve standard solutions containing DNC and d8-DNC in 1 methanol/water (75:25 v/v) were prepared at this time.

2A

  25 µl portion of the final sample extract was injected onto the LC-MS/MS system. The 3 LC-MS/MS system consisted of a Hewlett Packard (Stockport, Cheshire, UK) HPLC system, 4 comprising an 1100 Series binary pump, autosampler and solvent degasser, was coupled via an 5 electrospray interface to a Quattro LC (Micromass, Wythenshawe, UK), which operated in negative 6 ion mode. The [M-H] -ion at m/z 301 was monitored along with two transition ions at m/z 137 and 7 107 for DNC and the [M-H] -ion at m/z 309 for the internal standard, d8-DNC. The LC column used 8 was a Luna 5 µm C18 (2) 250 × 4.6 mm (Phenomenex, Macclesfield, Cheshire, UK). The mobile 9

81

  system consisted of a model 600 HPLC pump with a model 717 autosampler and model 484 UV 9 detector (set at 350 nm), all from Waters (Milford, MA, USA). The separation was carried out on a 10 stainless-steel analytical column (250 × 4.6 mm i.d) equipped with a Securiguard™ pre-column, 11 both packed with Hypersil BDS C 18 material (Phenomenex, Cheshire, UK). The column 12 temperature was maintained at 40°C. The mobile phase, consisting of water/acetonitrile (55:45, 13 v/v), was pumped at 1 ml/min. Under these conditions the retention time of DNC was 14 approximately 13 min. A Waters 746 data processing module was used for recording and 15 by fortifying negative control egg and liver samples at concentrations of 21 0, 50, 100, 250, 500 and 1000 µg kg -1 and muscle at concentrations of 0, 1, 2.5, 5, 10, 25 and 50 22 µg kg -1 of DNC for calibration. Calibration curves were prepared by plotting response as a function 23 of DNC concentration (µg kg -1 ). The DNC concentration in test samples was read directly from the 24 calibration curve prepared from fortified samples that were run with that particular batch.Calibration curve standard solutions containing DNC and d-8 DNC were prepared in 2 methanol/water (75:25 v/v). The DNC standards were prepared at the concentration 0, 100, 200, 3 300, 400 and 500 µg kg -1 standard equivalent along with known equal amount of internal standard 4 d-8 DNC. The calibration curve was obtained by plotting the standard concentrations against the 5 peak area ratios of d-8 DNC and DNC. The DNC concentration in unknown samples was reported 6 as internal standard corrected value.Standards were prepared at concentrations of 0, 50, 100, 250, 500, 1000 and 2000 ng ml -1 in 10 acetonitrile/water (80:20, v/v). Calibration curves were prepared by plotting peak area as a function 11 of DNC concentration (ng ml -1 ). The DNC concentration in test samples was determined from the 12 peak areas obtained for test sample extracts, as calculated from the calibration curve. The DNC 13 concentration in test samples was reported corrected for recovery using recovery factors 14 calculated from fortified samples that were run with that particular batch.15 16Validation of methods17The methods used in this comparison were validated according to current EU guidelines as 18 described in Commission Decision 2002/657/EC. The assays were validated to allow detection or 19 determination of DNC to an MRL of 200 µg kg -1 . The sensitivity of the Biacore TM biosensor, HPLC-20 UV and LC-MS/MS assays are much lower than the MRL. Limits of determination of DNC are 33.2, 21 12.5 and 10 µg kg -1 in liver; 34.8, 12.5 and 10 µg kg -1 in egg; and 5, 5 and 2 µg kg -1 in muscle, Liver samples (n = 736) were taken from 16 poultry companies (coded A to P), which were 1 representative of over 95% of domestic broiler production on the island of Ireland. In 2002, 2 samples were collected from companies A to H between March and November; no samples were 3 taken during May. The majority of the 238 samples were collected during the months of September 4 (n = 52), October (n = 89) and November (n = 81). In 2003, samples were collected from 5 companies A to H between February and November. The lowest and highest number of samples 6 were taken during the months of June (n = 7) and April (n = 46), respectively. Samples were 7 collected from companies I to P between May 2002 and April 2003. A total of 265 samples were 8 collected and the monthly sampling numbers are described in Figure 2. Samples were screened 9 using the immunobiosensor assay and samples found to contain DNC at levels greater than 33 µg 10 kg -1 were selected for confirmatory analysis. (n = 342) were taken from 13 poultry companies. Three of the companies 14 involved in the liver survey (A, G and H) did not participate in this study. Samples were collected 15 from companies B to F (n = 127) between July and December 2003. A total of 215 samples were 16 collected from companies I to P between May 2003 and April 2004 at a frequency of 16 to 2117 samples per month. Samples were screened using the immunobiosensor assay and samples 18 found to contain DNC at levels greater than 5 µg kg -1 were selected for confirmatory analysis.

  imported broiler breast muscle was undertaken in the period 2003 to 2004. In total, 217 2 samples of imported breast muscle were obtained from retail outlets, from Environmental Health 3 Officers carrying out inspections at food premises, by official agriculture inspectors at border 4 inspection posts and meat processing companies. Samples were screened using the 5 immunobiosensor assay and samples found to contain DNC at levels greater than 5 µg kg -1 were 6 selected for confirmatory analysis.

  muscle, compared with liver, is supported by the pharmacokinetic profiles, which 1

6 7

 6 Seasonal variation in levels of DNC in muscle 8The seasonal variation for DNC residues in breast meat is shown in Figure4. The average monthly 9 incidence of muscle samples containing DNC is 20%. The highest incidence occurred in the 10 months of May, November and March with 28, 28 and 42% of muscle samples containing DNC 11 residues, respectively. The proportion of liver samples likely to contain DNC at levels >200 µg kg -1 12 was estimated from the muscle survey results. A level of approximately 7 µg kg -1 DNC in muscle 13 has been estimated as equivalent to approximately 200 µg kg -1 in liver tissue. This approximation 14 is derived from the mean values obtained for a comparison of DNC residues in liver and muscle for 15 birds raised in deep litter (9.0 µg kg -1 muscle ≈ 200 µg kg -1 liver) and those on wire flooring (5.5 µg 16 kg -1 muscle ≈200 µg kg -1 liver) [Cannavan and Kennedy 2000]. Using this estimation, the 17

  levels in muscle sampled from different companies 22 The breakdown by company of muscle samples containing DNC residues and corresponding liver 23 samples (estimated as containing DNC residues >200 µg kg -1 ) is shown in Table I. No samples 24 from companies E and F contained measurable DNC residues and company C had only 3% of 25 samples containing measurable DNC residues. For the other companies, the proportion of muscle 26 samples containing measurable DNC ranged between 20 and 50%. A comparison between the 27 liver survey (2002/03) and estimated liver survey (2003/04) shows that for some companies (B, I,

5

  2002/03 and estimated for 2003/04) may vary between companies and within companies from year 6 to year. The results of the surveys show that DNC residues in broilers occur throughout the year 7 and that flocks require frequent sampling (such as each month), proportional to scale of 8 production, to identify the situation. 217 samples of imported breast meat were sampled over the period May 2003 to 12 September 2004. The objective of the survey was to make a comparison between DNC residues in 13 domestic and imported poultry meat consumed on the island of Ireland. The results of the survey 14 are shown in Table II. Samples were sourced from wholesalers, meat processing companies, 15 border inspection posts and retail outlets. The imported breast meat samples originated from 11 16 different countries. DNC residues were determined in 13 samples at levels ranging from 2.7 to 18.7 17 µg kg -1 . The results of the survey indicate that DNC residues were at a lower level in imported 18 meat (6%) compared to domestic meat samples (26%). Samples from only three countries had 19 measurable DNC residues (France, Thailand and The Netherlands). The relatively low number of 20 positives for DNC in imported meat suggests (a) that alternative anticoccidial agents may be used 21 in other countries and/or (b) practices are adopted at feed mills and on farms that allow better 22 control of DNC residues in poultry production. of table eggs was carried out during 2003 to 2004. A total of 546 egg samples were 26 collected and DNC residues were determined in nine samples. No residues were detectable (i.e.27not detected above 34.8 µg kg -1 by immunobiosensor and/or confirmed above 12.5 µg kg -1 using

  The results for DNC residues in poultry liver and eggs samples between 2002 and 2005 indicate 1 that the percentage of liver samples containing DNC at levels ≥200 µg/kg decreased to 7% in 2005 2 (O'Keeffe et al. 2005; O'Keeffe et al. 2006). This was a 50% reduction in positives from the 3 previous three years even though the useage of Maxiban throughout Ireland had increased. The 4 results of official food inspection for 2006 will provide a clearer indication if this reduction in DNC 5 positives is sustainable or just a brief improvement. The reduction in DNC positives can be largely 6 attributed to on-farm investigations, which were carried out with the aim of identifying the cause of 7 DNC residues during 2004 to 2005 (O'Keeffe et al. 2007). In addition, there has been increased 8 scrutiny placed on the control of DNC residues by regulatory agencies. As a result, a number of 9 companies have instigated their own testing programs, which are aimed at fulfilling self-monitoring 10 criteria. The results for egg testing between 2002 and 2005 indicate that the percentage of DNC 11 positives in eggs remains static at approximately 1.5%. frequent contaminant of poultry products on the island of Ireland and has been shown to 16 be a contaminant both of liver and breast meat. Residues may also occur in eggs but are typically 17 at less than 2% of samples. Residues in eggs may be largely attributed to contaminated feed 18 because administration of nicarbazin to layers is not approved because it affects bird fertility and 19 egg shells. DNC residues occur in poultry samples throughout the year, indicating that this 20 anticoccidial needs to be monitored continuously in poultry. Residues in liver and meat can be 21 caused by a number of different factors, but may be attributed mainly as due to improper feed 22 management on farms and at feed mills (O'Keeffe et al. 2007). The number of liver samples 23 exceeding the MRL of 200 µg kg -1 DNC may vary considerably within individual companies 24 between years. In 2005, there was a reduction in the number of broiler liver samples containing 25 DNC at >200 µg kg -1 , to approximately 7% of samples tested in the monitoring programmes /05 with the aim of identifying the causes for DNC residues in poultry (O'Keeffe et 1 al. 2007). In addition, there has been increased scrutiny on the control of DNC residues in poultry 2 by regulatory authorities and a number of companies have instigated there own testing 3 programmes to fulfil self-monitoring as required by EU food law (96/23/EC). 1999, Freedom of information summary -Combined use ofMAXIBAN and BMD in Chicken

58

  ., Danaher, M.,Anastasio, A., Cortesi M. L. and O'Keeffe, M., 2005, Efficient HPLC ., Fodey, T., Crooks, S.R.H., Delahaut, P. and Elliott, C.T., 2002, The production and 2 characterisation of dinitrocarbanilide antibodies raised using antigen mimics. Journal of 3 Immunogical Methods. 264, 45-51.4De Giovanni, F., Amendola, F., and Cortesi, M.L., 1989, Residui di nicarbazina in uova del 6 marcato. Industrie Alimentari, 28, 10-13. 7 Gallo, P., Serpe, L, 1997, Monitorie dei Residui Di Nicarbazina in Matricia do originie animale 9 biennio 1994-1996. Industrie Alimentari, 6,18 -620. 10 11 McCarney, B., Traynor, I. M., Fodey, T.L., Crooks, S. R. H., Elliott, C.T., 2003, Surface Plasmon 12 resonance biosensor screening of poultry liver and eggs for nicarbazin residues. Analytica Chimica 13 Acta. 483, 165-169. 14 15 McEvoy, J.D.G., Smyth, W.G. and Kennedy, D.G., 2003, Contamination of animal feedingstuffs 16 with nicarbazin: investigations in a feed mill. Food Additives and Contaminants, 20, 136-140. 17 18 Oishi, Y., and Oda, T., 1989, Studies on residues of nicarabazin in eggs of laying hens. Shokuhon 19 Eiseigaku Zasshi, 30, 542-547. 20 21 O'Keeffe, M. and Coen, K., 2005. National Food Residue Database Report 2005, Teagasc, Dublin, , M., Rehmann, F.J. and Coen, K., 2006. National Food Residue Database Report 2006,

Figure 1 Figure 2 Figure 3 Figure 4

 1234 Figure1Levels of DNC residues in broiler liver samples(n = 736)

8

  Feeds Approval Date: January 4 1999 (http://www.fda.gov/cvm/efoi/section2/140-926.pdf, Official Journal of the European-Commission Regulation No. 657/2002/EC. No. L Cannavan, A. and Kennedy, D.G., 2000, Possible causes of nicarbazin residues in chicken tissues.

	9
	10	accessed 3/11/2004).
	11
	12	Anon., 2001, Official Journal of the European-Commission Regulation No. 2205/2001/EC. No. L
	13	297/3.
	14
	Anon., 2002, 15
	16	221/8.
	17
	18	Anon., 2007, Community Register of Feed Additives pursuant to Regulation (EC) No 1831/2003
	19	Appendixes 3 & 4. Annex : List of additives Rev. 16 (June 2007).
	20
	21
	22	Food Additives and Contaminants, 17, 1001-1006.
	23
	24	Cannavan, A., Ball, G. and Kennedy, D.G., 2000, Nicarbazin contamination in feeds as a cause of
		residues in eggs. Food Additives and Contaminants, 17, 829-836.

Table I

 I Variation in the percentage of liver and muscle samples containing DNC residues that were surveyed from companies A to PTable II The incidence of DNC residues in breast muscle samples imported into the island of Ireland

	Page 21 of 25				Food Additives and Contaminants Food Additives and Contaminants	Page 20 of 25
				14
				11.8%
				12
	Country			Liver Survey (n = 736) Number of samples tested Domestic Muscle Survey (n = 342) Number of samples
	Company	Samples containing DNC ≥12.5 µg kg -1 (%) 6 8 10	Samples containing DNC ≥200 µg kg -1 (%) 8.8%	7.7%	Samples containing DNC ≥5 µg kg -1 (%) >5 µg/kg Estimate of liver samples containing (%) DNC ≥200 µg kg -1 7.2% containing DNC at
	A The Netherlands B C Denmark	84 47 15 2 4		26 16 1	68 26		-38 3	2.3%	4 0	-29 0 2.6%
	D E F G H I Belgium France Brazil Chile	41 22 0 0 40 46 o F r 0 F r o 12.5-49	13 0 0 0 33 11 50-99	36 0 0 --36 100-199 200-499 10 7 58 2	0 8 0 0 500-999	14 0 0 --17 >1000
	J K L M N O P Germany Thailand Romania New Zealand	67 83 33 25 44 63 59 P e e 25 25 0 6 8 42 16 r P e e r	14 22 5 4		30 22 27 20 20 50 20	0 1 0 0	33 9 25 17 3 29 8
	Mean UK			42		14 R 1 R		23	0	14
						e e
							v i e v i e
								w w
									O n l O n l
									y y

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Cannavan et al. 2000, McEvoy et al. 2003], withdrawal period [Cannavan and Kennedy 2000] and

reconstituted in cyclohexane (1 ml) by vortexing (15 sec). An aliquot (250 µl) of methanol/water

(75:25, v/v) was added, the tubes were vortexed (10 sec) and placed in a water bath (37°C, 10

min) prior to centrifugation (1200×g, 10 min). Aliquots (200 µl) of the aqueous layer were removed

DNC in muscle tissue. Muscle samples (5 g) were weighed into 30 ml polypropylene tubes and

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reconstituted in acetonitrile/water (70:30, v/v, 500 µl). Samples extracts were passed through C 18

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(O'Keeffe et al. 2006, Shortt 2006). The available results from official food inspections for 2006

indicate a similar lower proportion of samples containing DNC residues at levels >200 µg kg -1 . The

reduction in DNC positives may be attributed partly to on-farm investigations, which were carried

Acknowledgements

The authors would like to acknowledge safefood, the Food Safety Promotion Board, for funding 2 this project. The authors would also like to acknowledge the assistance of Agriculture Inspectors