Keith Scudamore

Sue Patel

Keith ; Scudamore

Mycotoxins Kas

Keith A Scudamore

Occurrence of fusarium mycotoxins in maize imported into the United Kingdom, 2004-7

Keywords: Mycotoxins -fumonisins, Mycotoxins -fusarium, Mycotoxinstrichothecenes, Mycotoxins -zearalenone Food Types: Cereals, Cereals and grain deoxynivalenol, zearalenone, fumonisins, nivalenol, cleaning, maize 38 39

This study examined a total of 82 consignments of French and Argentinean raw maize 12 as received at maize mills in the UK during the period 2004-2007. Samples were 13 analysed for deoxynivalenol (DON), nivalenol (NIV), other trichothecenes, 14 zearalenone (ZON) and fumonisins B 1 , B 2 , and B 3 (FB 1 , FB 2 , and FB 3 ) using fully 15 validated analytical methods with limits of quantification of 10 µg kg -1 for DON,NIV 16 and each fumonisin mycotoxin and 3 µg kg -1 for ZON. All samples except two 17 containing fumonisins met the EC statutory maximum permissible levels for DON,18 ZON and FB 1 + FB 2 as operating in 2007. The maximum concentrations of DON,19 NIV, ZON and FB 1 + FB 2 , found were 444, 496, 165 and 5002 µg kg -1 respectively. 20 Fumonisins were detected in almost every sample with 65% of Argentinean maize 21 containing more than 1000 µg kg -1 of FB 1 + FB 2 . In contrast ZON was not detectable 22 in almost 50% of consignments. During this period there was a distinct difference in 23 mycotoxin concentrations between harvests and geographic origin. Flint maize from 24 Argentina usually contained lower concentrations of DON and related trichothecenes 25 and higher levels of fumonisins than maize from France, although concentrations of 26 fumonisins up to 2000 µg kg -1 or greater occurred in samples from both regions. The 27 incidence and concentrations of fumonisins were similar to those in a similar previous 28 survey while zearalenone concentrations were lower. The distribution of mycotoxins 29 in multi-hold ships was also investigated showing that fumonisins were much more 30 evenly distributed than DON thus indicating their general level in the ship as a whole. 31

The effect of cleaning regimes was found to be very variable, especially for DON,32 ranging from no removal of mycotoxins to >50% in some instances but was not 33 related to concentration. Evidence here suggests that while cleaning is essential for 34 removing foreign bodies prior to milling it cannot be used as a reliable tool for 35 reducing mycotoxins. 36

Introduction 40

The risk to human health posed by mycotoxins has been assessed by international 41 organisations including WHO/FAO and the European Commission and as a result 42 maximum permissible limits for certain mycotoxins have been introduced. Limits 43 have been set within the EU for DON and ZON in cereals including wheat, maize, 44 oats and for fumonisins in maize (EC 2006a(EC , 2007) ) and are under consideration for 45 T2-toxin and HT2-toxin. A number of mycotoxins occur in maize often in 46 combination and include aflatoxins (Andersen et al. 1975), ochratoxin A (Shotwell et 47 al. 1969) and a number of fusarium mycotoxins, DON [START_REF] Gilbert | A survey of the occurrence of the 419 trichothecene mycotoxin deoxynivalenol (vomitoxin) in United Kingdom 420 grown barley and in imported maize by combined gas chromatography-mass 421 spectrometry[END_REF], NIV and 48 other trichothecenes (Vesonder et al. 1973), ZON [START_REF] Shotwell | Survey of corn for 453 aflatoxin, zearalenone, and ochratoxin[END_REF], fumonisins 49 [START_REF] Gelderblom | Fumonisins -novel mycotoxins with cancer-promoting 416 activity produced by Fusarium moniliforme[END_REF], moniliformin [START_REF] Sharman | A Survey of the occurrence of the 443 mycotoxin moniliformin cereal samples from sources world-wide[END_REF], Scudamore et al. 1997, 50 1998) and beauvaricin, fusaproliferin and fumaric acid [START_REF] Ritieni | Occurrence of Fusaproliferin, Fumonisin B 1 , and 432 Beauvericin in Maize from Italy[END_REF] Maize for human food entering the UK comes principally from France and Argentina 62 and is purchased on contract requiring consignments to meet specified standards 63 (including those for mycotoxin concentrations) laid down by the buyer, usually equal 64 to or lower than regulatory limits. Mycotoxin levels may thus not be entirely 65 representative of the mycotoxin concentrations occurring in the countries where the 66 maize is grown but does represent that imported and used by UK Industry. In the 67 earlier study of maize entering the UK, consignments were examined for aflatoxins, 68 ochratoxin A, fumonisins and ZON but did not include DON. 69 70 Fumonisins first reported in 1988 have been shown to occur widely, but mainly only 71 in maize [START_REF] Shephard | World-wide survey 450 of fumonisin contamination of corn and corn--based products[END_REF]. Fumonisins, ZON, DON, NIV and other 72 trichothecene mycotoxins are produced by different species of Fusarium so that 73 occurrence of a mixture of mycotoxins usually indicates that the maize has been 74

infected by more than one fusarium species. The human consumer is not exposed 75 directly to the quantities of mycotoxins in the raw maize but to the mycotoxins 76 remaining in the food products manufactured after milling and processing of the raw 77 cereal. As maize processing is a complex technology (e.g. [START_REF] Kent | Kents Technology of Cereals, 4 th Edition[END_REF] the 78 levels of mycotoxins in the finished product may not reflect the concentrations in raw 79 maize as the amounts surviving through the food processing chain will depend on the 80 properties of the mycotoxin and on the nature of the process(es). 81 82 Reliable information about the frequency and concentrations of mycotoxins occurring 83 in maize are required, including trends over the longer term and this survey thus 84 aimed to examine the concentrations of fusarium mycotoxins in raw maize entering 85 the UK from the harvests in the period 2004 to 2007 and to compare the results where 86

Sampling and analysis 91

Maize for human consumption in the UK is imported in bulk consignments by boat 92 and then either transferred directly via conveyer belt to dockside mills or transported 93 by truck. Maize from France arrives in vessels usually holding 2-6000 tonnes and that 94 from Argentina in larger consignments of 20-30,000 tonnes that may be contained in 95 up to 10 or more holds although not all holds will contain maize and a range of other 96 imports are likely to be carried simultaneously. Samples for this study were supplied 97 by three large UK millers. 98 99

Official sampling procedures 100

Introduction of statutory limits for fusarium mycotoxins were accompanied by 101 procedures for sampling and for analysis when inspecting for enforcement purposes 102 (EC 2006b). It is however accepted that it may be appropriate to use other sampling 103 methods and procedures for purposes other than for official control. Operation of 104 those sampling procedures here would be both onerous and costly for industry and as 105 sampling was not intended for enforcement purposes, 'alternative' procedures were 106 devised based as far as possible on existing in-house practice in order to disrupt 107 everyday production as little as possible. Samples collected were kept cool and dry out 108 All three millers have in-house sampling protocols at in-take to their mills although 112 each procedure is different. Each company supplied samples based on the internal 113 quality control system in place, or adaptations thereof. 114 115 Company 1 supplied 30 consignments of raw maize over a 3-year period. Maize was 116 transported from ship to mill in vehicles each containing 28 tonnes. In previous 117 studies from this mill, 20 consecutive lorries were sampled (Scudamore and Patel, 118 2000). In this study one vehicle was isolated on each occasion and 30 incremental 119 samples were taken and bulked to give a 10kg sample for analysis. In further studies a 120 28 tonne load was cleaned and the maize milled to obtain representative samples of 121 the product streams, which included grits, flour and other ingredients.

Analytical methods 139

In total 82 different ship consignments of whole in-take maize (together with some 140 cleaned samples and milled samples) were examined over an approximate 3-year period 141 spanning four different harvest years. The quantity of sample taken as described earlier 142 was 4 to 10 kg depending on the agreed method of sampling used and a sub sample was 143 sent for analysis. On receipt, the whole sample was ground to pass through a 0.8 mm 144 screen and mixed for 30 minutes in a Gardner horizontal mixer to ensure homogeneity. 145

Samples were stored at -20°C if not analysed immediately. The spike level for DON, fumonisins and for ZON were 400, 400 and 50 µg kg -1 160 respectively. When results were significantly above these levels, they were repeated and 161 spiked at a level appropriate for each toxin. 162 163 The limit of detection and limit of quantification respectively for each mycotoxin were 164 5 µg kg -1 and 10 µg kg -1 for DON, the other trichothecenes and each fumonisin 165 mycotoxin and 1.5 µg kg -1 and 3 µg kg -1 respectively for ZON. The limit of detection 166 is defined as 3 times the electronic baseline noise and the limit of quantification as 6 167 times baseline noise. Calibration curves for each mycotoxin were plotted with the 168 lowest calibration points respectively being equivalent to 10, 3 and 10 µg kg -1 for 169 DON, ZON and fumonisins. After analysis samples were retained and stored at -170

20°C. 171 172

Analysis 173 DON together with the other trichothecene mycotoxins was determined following the 174 method of Patel et al. (1996). ZON was determined using HPLC with fluorescence 175 detection [START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, ZON and 438 fumonisins in maize imported into the United Kingdom[END_REF]. The method for fumonisins was a modification 176 of the method originally published by Shephard et al. (1990) and modified (Scudamore 177 and Patel, 2000). contained between 1000 µg kg -1 and 3999 µg kg -1 although this was much less than the 210 97 % of total fumonisins found in this range in the survey of 2000 although that study 211 only covered a 10-month period. Levels of DON and ZON in the current study were 212 much lower rarely occurring at more than 10 % of the statutory limits while a level of 213 496 µg/kg of NIV was found in one consignment. However, while DON was detected 214 in every sample ZON was undetected in 50 % of the consignments. NIV occurred in 215 65 % of samples, although the mean value of 57 µg kg -1 resulted from a heavily 216 skewed distribution where a few consignments contained relatively high 217 concentrations so that the median value was only 13 µg kg -1 . In the earlier study ZON 218 was greater than 20 µg kg -1 in every consignment with a maximum value of 584 µg 219 kg -1 (cf. 42 µg kg -1 here). 220

INSERT: TABLE 2 221 222

Results for French consignments of maize are given in Table 2 and maize from different sources and seasons as this will be affected by physical treatment 259 of these grain consignments before shipping selection before export to ensure it will 260 meet the specifications of the buyer. Constant monitoring of maize is required at 261 source before export to avoid the arrival of a ship containing maize with unacceptable 262 levels of mycotoxins (e.g. in excess of regulatory limits) as this would represent a 263 huge problem and a potential financial disaster. 264 265

Relationship between fumonisin mycotoxins 266

Legislation for fumonisins is for the combined concentration of FB 1 + FB 2 . Melcion 267 et al. (1997), studied the biosynthesis of fumonisins in culture by various strains of 268 Fusarium moniliforme. Some strains only produced FB 1 while others varied widely in 269 the amounts and proportions of the various analogues obtained. Rheeder et al. (2002) 270 reported that FB 1 typically accounts for 70 to 80 % of the total fumonisins produced, 271 while FB 2 usually makes up 15 to 25 % and FB 3 usually makes up from 3 to 8 % when 272 cultured on corn or rice or in liquid medium. 273

INSERT: TABLE 4 274

The relationship between levels of FB 1 FB 2 and FB 3 for all samples of intake maize is 275 given in Table 4 as the mean ratios of FB 1 to FB 2 and FB 1 to FB 3 in the three areas 276 from which the maize originates and is similar irrespective of origin. Combining all 277 results gives a ratio of 3.6 for FB 1 to FB 2 with a SD of 23 % On the basis of these 278 results it is suggested that results found for FB 1 allows extrapolation to a working 279 value for FB 1 + FB 2 . 280 281 During this work the opportunity arose to obtain samples from different holds in a 283 single ship as there appears little or no information about how the distribution of 284 mycotoxins vary in such a situation. This was carried out in 5 ships in which the 285 maize from either 6 or 7 different holds each containing several thousand tonnes of 286 maize could be examined. Each ship was loaded with maize from different suppliers 287 from Argentina, but all originating from the same general area. 288

Variation in mycotoxin concentrations in different holds of multi-hold ships 282

INSERT: TABLE 5 289

The distribution of mycotoxins between holds in 5 different vessels is given in table 5. 290

The variability between concentrations from the different holds indicated by % SD 291 between samples showed that the % SD for fumonisin values were usually much 292 lower than those for DON, ZON and NIV confirming the visual picture shown in 293 Figure 1. Despite the variability observed, the maximum value for DON, ZON and 294 NIV from any individual hold was still never more than 3 times that of the mean 295 concentration of maize in the ship. The maximum value for FB 1 + FB 2 was only 1.7 296 times the mean in vessel E and this value was lower in the other vessels. No sample in 297 any hold exceeded the EC statutory limits set for fumonisins despite their relatively 298 high mean concentration, for example, in ship B being more than 50 % of the statutory 299 limit. 300 301

Effect of cleaning 302

Maize on intake is subjective to a comprehensive cleaning process that removes 303 stones, metal objects cobs and other foreign bodies. A succession of sieves, aspirators 304 and separation systems will then remove broken maize seeds and dust. The 305 effectiveness of cleaning for removing mycotoxins from maize has been reported 306 previously as being very variable. [START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, ZON and 438 fumonisins in maize imported into the United Kingdom[END_REF] studied the effect of 307 cleaning on the levels of aflatoxins, zearalenone and fumonisins in maize in a large 308 commercial mill. Aflatoxin and fumonisin concentrations were reduced by about 30 -309 40 % but little reduction was observed in the levels of zearalenone. No significant 310 reduction of aflatoxin was observed earlier by [START_REF] Brekke | Cleaning trials for corn containing 401 aflatoxin[END_REF] while cleaning also 311 appears to be relatively ineffective for deoxynivalenol [START_REF] Scott | Distribution 435 of the trichothecene mycotoxin deoxynivalenol (vomitoxin) in hard red spring 436 wheat[END_REF]Abbas et al., 312 1985;[START_REF] Seitz | Distribution 441 of deoxynivalenol in soft wheat mill streams[END_REF]. 313

INSERT: TABLE 6 314 315

INSERT: TABLE 7 316

Tables 6 to 8 present results showing the effect of cleaning on mycotoxin 317 concentrations in the 3 mills. Where analytical results for a matched pair of raw maize 318 and clean maize are available the apparent reduction or change in mycotoxin 319 concentration has been calculated for each mycotoxin. The mean change in 320 concentration achieved for each mycotoxin for all samples was then calculated and 321 these values are given in the tables. The changes achieved in mills 1, 2 and 3 were +5 322 %, 28 % and -1 % respectively for DON, + 11, + 23 % but -54% for ZON and + 4 %, 323 + 18 % and -48 % for FB 1. Results for FB 2 and for FB 3 were similar to those for FB 1 324 within each mill. 325

INSERT: TABLE 8 326

The concentrations of mycotoxins in the consignments of maize studied are 327 also given in the tables but these do not appear to influence the degree of cleaning 328 achieved.. Despite the difference in scale, the principles of operation were similar. 329

Other reports of maize cleaning at the industrial scale show similar variability and so 330 the effectiveness of cleaning is probably more related to the condition of the grain 331 consignment than to the specific mill involved. The effect varied from being 332 ineffective on some occasions to substantially reducing mycotoxins in others with up 333 to 80 % of the mycotoxins being removed at the extreme. The effects on DON 334 appeared to be extremely variable although this is most likely to be an artefact of 335 sampling. 336 337

Occurrence of other Fusarium mycotoxins 338

The only mycotoxins detected in intake maize other than DON, ZON, NIV and 339 fumonisins were low levels of 3-ac DON, 15-ac DON, fusarenone-X, HT2 and T2 340 although T2 and HT2 were never detected here in Argentinean maize. When these 341 trace concentrations were found in intake maize, they could usually be confirmed after 342 milling by the higher levels in the milling streams such as bran, meal and germ in 343 which mycotoxins are concentrated (results to be reported elsewhere). 344 345

Conclusions 346

One or more fusarium-derived mycotoxins were present in quantitative amounts in 347 every sample of raw maize examined. Total fumonisin concentrations were frequently 348 greater than 1000 µg kg -1 , but since legislation for fumonisins was introduced, no 349 consignment contained FB 1 +FB 2 above the statutory limit of 4000 µg kg -1 while no 350 concentrations of DON, ZON and NIV exceeded 500 µg kg -1 , 200 µg kg -1 and 500 µg 351 kg -1 respectively. Mycotoxins levels varied between source and season. Sampling from 352 different holds in large ship consignments suggested that variation in the 353 concentrations of mycotoxins in the vessel tended to be less for fumonisins than for 354 Information about the fate of mycotoxins during processing is required to assist with 357 the estimation of daily intake of mycotoxins from maize products and relate these with 358 the levels in raw maize. The acquisition of these data should be encouraged to ensure 359 that they are available for any discussion relating to further EU legislation for 360 mycotoxins in raw cereals and cereal-based foods. 361 362 Sampling according to the methods proscribed by the EC Regulations for enforcement 363 purposes is difficult, time consuming and not always practically possible but 364 alternative samples for quality control may be suitable. The UK Food Standards 365 Agency has recommended guidelines to help clarify sampling requirements for use by 366 enforcers and trade. 367 368 Despite severe problems of very high concentrations of fumonisins reported from 369 Europe in maize from the 2006 harvest, UK maize millers have been able to import 370 maize that in most instances has mycotoxin levels significantly below the current 371 statutory limits. Should it become agronomically and economically feasible to grow 372 maize with the appropriate characteristics needed for products for human consumption 373 in the UK, it could be anticipated that concentrations of fumonisins would be low 374 compared to those in maize from hotter parts of Europe although frequency and levels 375 of DON and ZON might be higher. 376 377 Cleaning tends to reduce mycotoxin concentrations somewhat, but the results obtained 378 are very variable and unpredictable. However it is concluded here that the cleaning 379 operations are essential for removing foreign bodies prior to milling rather than as a 380 tool for reducing mycotoxins. 381

  samples from 26 consignments of raw maize over a 3-year 124 period. Sampling was by autosampler from trucks of approximately 20 tonne capacity 125 resulting in 60 incremental samples each of approximately 100 g giving a bulk sample 126 of 6 kg for analysis. Each spearing represented a combination of samples from top, 127 middle and bottom of the truck. 128 129 Company 3 supplied samples from 26 ship deliveries of maize over a 3-year period. 130 Samples of maize from multiple holds of these ships were in addition taken on six 131 occasions. Representative intake maize samples were collected from the ship to mill 132 conveyer belt approximately hourly during a continuous eight-hour period 133 representing a working day run. Eight samples of about 0.5 kg were scooped from the 134 flowing maize streams to provide a composite of about 4 kg of material. Because of 135 of the consignments unloading takes several days and is done hold by 136 hold. The 4 kg bulk sample was thoroughly mixed and sent for analysis. 137 138

  used were UKAS (ISO 17025) accredited. All reagents were 148 analytical grade and all solvents were of HPLC grade. Mycotoxin standards of DON, 149 NIV, other trichothecenes, ZON and fumonisin B 1 , B 2 and B 3 , were purchased from 150 commercial sources. Trichothecene and ZON standards were quantified prior to use by 151 UV spectrometry. 152 153 All analyses were conducted with a spiked sample, i.e. a known amount of toxin was 154 added to a sample of ground maize each day prior to extraction, clean-up and HPLC 155determination for each batch of 1-5 samples. These results were used to assess 156 recovery and all reported results were corrected using the values obtained. Recoveries 157 in the range 70-110% were considered acceptable.

  are presented 223 separately for the 2 mills and combined to give the means for all French consignments 224 to enable comparison with the Argentinean maize. The maize used by the 2 French 225 mills originates from different growing areas. The concentrations of mycotoxins in 226 maize imported by Mill 2 were somewhat higher than those from Mill 1 (except for 227 NIV) although this could merely reflect different purchasing quality specifications set 228 by the two Companies rather than climate or other factors. Concentrations of DON 229 and ZON were higher on average in French maize than in the Argentine maize while 230 fumonisin concentrations were lower. Mean concentrations of NIV were quite similar The reduction of ZON (and probably DON) concentrations since 2000 may in part be 234 due to the introduction of limits for these mycotoxins that were introduced in 2006 235 while limits for fumonisins were not introduced until 2007. 236 237Previous studies of French maize indicated that amounts of fumonisins (and 238 aflatoxins) decreased with higher latitudes, probably reflecting the cooler climate 239[START_REF] Scudamore | Survey for aflatoxins, ochratoxin A, ZON and 438 fumonisins in maize imported into the United Kingdom[END_REF]. Fumonisin levels (and aflatoxin concentrations) were 240 higher in Argentinean maize. In contrast, there was no clear trend in the results for 241 ZON. This was suggested as reflecting the relative climate in the regions and is 242 consistent with the conditions necessary for the growth of the fungi responsible for the the results separated into the relative harvest years2004 to 2007. 247 Although the relative number of samples is small and is not the same for each season, 248 the quantity of maize represented by the samples is high and should give a good 249 indication of the relative levels of mycotoxins in these years. Hence fumonisins 250 occurred at their highest levels by far in 2004 in maize from Argentina that suffered a 251 prolonged drought prior to harvest, and in maize from France in 2006, a year in which 252 fumonisins reached very high levels in parts of Europe during a very hot, dry summer. 253 DON, ZON and NIV were all at their lowest levels in the 2005 harvests in maize from 254 both Argentina and France. 255INSERT: TABLE 3 256Although this survey indicates the concentrations of DON, ZON and fumonisins in 257 imported raw maize over the years 2004-2007, care should be exercised in comparing 258

  this survey provide important information on the occurrence 383 and distribution of DON, ZON, NIV and fumonisins in maize. This information is 384 relevant both to the UK and to the EC and these data give a valuable indication of the 385 effectiveness of recently introduced EC limits for these mycotoxins. 386 387 Acknowledgements 388 These studies were funded by the UK DEFRA, Food Quality and Safety LINK 389 Programme and the UK Food Standards Agency and would not have been possible 390 without the enthusiastic cooperation and assistance from the staff of the three UK Hesseltine CW, Goulden ML. 1969. Ochratoxin A: Occurrence as a 455 natural contaminant of a corn sample. Applied Microbiology 17:765-766. 456 Vesonder RF, Ciegler A, Jensen AH. 1973. Isolation of the emetic principle from 457 Fusarium-infected corn. Contaminated with Aspergillus versicolor.

Table 1 :

 1 Occurrence of mycotoxins in raw maize fromArgentina, 2004Argentina, -2007 harvests 461 harvests 461

	No. of

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	470
	471
	472	Table 2 : Mycotoxins in raw maize from France ( UK intake mills 1 and 2), 2004-2007
		No. of				Mycotoxin, µg kg -1 (EC maximum)
		Consignments Parameter	(1750) DON	(350) ZON	FB 1	F B 2	FB3	(4000) FB 1 + FB 2	NIV
		30	Mean, mill 1	139	16	320	83	49	403	39
			Median, mill 1	41	<3	289	59	31	362	27
			Range, mill 1	<10-444	<3-86	<10-1010	<10-269	<10-180	<10-1279	<10-134
		26	Mean, mill 2	270	37	541	155	89	696	31
			Median, mill 2	254	23	439	120	67	543	22
			Range, mill 2	19-932	<3-165	14-2590	<10-811	<10-501	19-3401	<10-170
		56	Mean, all	200	26	423	115	68	538	35
			Median, all	131	11	293	61	47	365	25
			<10	1	23	2	7	8	2	15
			10-99	24	31	9	25	38	7	37
			100-349	18	2	20	20	9	19	4
			350-999	13	0	18	4	1	21	0
			1000-3999	0	0	7	0	0	7	0
			>4000	0	0	0	0	0	0	0
	473

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	474
	475
	476
	477
	478	Table 3: Mean fusarium mycotoxin concentrations in maize from different harvest years as received in the UK
		Country				Mycotoxin, µg kg -1
		(consignments	Harvest year	DON	ZON	FB 1	FB 1 + FB 2	NIV
		)
		Argentina
		( 4)	2004	117	27	2714	3648	14
		( 8 )	2005	45	6	1095	1439	16
		( 9 )	2006	82	6	1455	1952	109
		( 5)	2007	147	26	693	906	64
		France
		(12)	2004	273	41	316	398	54
		(21)	2005	52	4	319	408	17
		(19)	2006	338	39	697	893	46
		(4)	2007	232	51	469	600	34
	479
	480
	481
	482
	483
	484

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							Food Additives and Contaminants	Page 24 of 29
	485
	486	Table 4: Mean ratios values for the concentrations of FB 1 to FB 2 and FB 1 to FB 3 in
	487		raw maize
			Mill 1 (French)	Mill 2 (French)	Mill 3 (Argentine)	ALL
			FB 1 : FB 2 FB 1 : FB 3 FB 1 : FB 2 FB 1 : FB 3 FB 1 : FB 2 FB 1 : FB 3 FB 1 : FB 2 FB 1 : FB 3
		Mean	3.9	7.0	3.7	6.5	3.1	6.1	3.6	6.7
		Median	4.0	6.7	3.7	6.0	3.2	5.4	3.7	6.0
	488	Range SD, %	2.5-6.1 20 F 4.5-16.3 38 o r	2.2-5.8 21	3.4-14.7 40	1.9-4.0 17	3.6-15.3 48	1.9-6.1 3.4-16.3 23 42
				P
				e
					e r
						R
						e
						v i e
							w
								O n l
									y

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Table 5 :

 5 Variation in mycotoxin concentrations in maize taken from different holds of pairs of raw and cleaned maize are included in which both values are equal to or above 496 the quantitative limit. These are the numbers of values used for the calculations.

	489
	490	ships from the Argentine
	Ship Ref.		Mycotoxin, µg kg -1
	(holds	parameter	DON	ZON	FB 1 + FB 2	NIV
	sampled)
	A (6)	Mean	38	4	1789	10
		Median	30	<3	1856	7
		Range	12-86	<3-15	1231-2459	<10-23
		SD, %	69	123	27	70
	B (6)	Mean	42	18	2058	108
		Median	48	16	2170	91
		Range	<10-69	6-40	1238-2488 <10-243
		SD, %	58	65	21	89
	C (7)	Mean	65	6	1653	91
		Median	65	4	1528	97
		Range	11-118	<3-17	1230-2506 <10-153
		SD, %	61	95	31	69
	D (7)	Mean	148	15	446	<10
		Median	137	13	414	<10
		Range	63-284	11-21	158-653	-
		SD, %	49	29	39	-
	E (6)	Mean	218	37	1383	46
		Median	134	17	1312	28
		Range	43-630	9-100	456-2365	<10-153
		SD, %	99	104	55	126
	SD, % mean of all	67	83	35	88
	491

Table 7 :

 7 Effect of cleaning on mycotoxin concentrations, mean of all pairs of raw and cleaned maize are included in which both values are equal to or above 504 the quantitative limit. These are the numbers of values used for the calculations.

	Page 27 of 29	Food Additives and Contaminants
	502
	503	consignments, mill 2
					Mycotoxin
			DON	ZON	FB 1	FB 2	FB 3
		Number of calculations †	9	6	8	7	7
		Mean change, %	+28	+23	+18	+43	+5
		Median change, %	+32	+27	+6	+3	+6
		Range mean change, % -51 to +276 -64 to +231 -30 to +247 -14 to +384 -25 to +137
		Range of concentrations*	42-743	14-130	14-1250	23-440	14-191
		% SD	55	63	47	75	23
	† only 505
	506	* µg kg -1 in raw maize
	507

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Table 8 :

 8 Effect of cleaning on mycotoxin concentrations, mean of all pairs of raw and cleaned maize are included in which both values are equal to or above 512 the quantitative limit. These are the numbers of values used for the calculations.

		Food Additives and Contaminants		Page 28 of 29
	510
	511	consignments, mill 3
					Mycotoxin
			DON	ZON	FB 1	FB 2	FB 3
		Number of calculations †	10	8	10	10	10
		Mean change, %	-1	-54	-48	-52	-51
		Median change, %	-11	-67	-57	-61	-66
		Range mean change, % -75 to +143 -74 to -12	-77 to 0	-84 to +3	-83 to +4
		Range of concentrations* 44-191	5-42	377-2740	128-1230	74-711
		% SD	5419	62	52	51	59
	† only 513
	514	* µg kg -1 in raw maize
	515

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Rainer Schuhmacher

Occurrence of deoxynivalenol and its 3-β-D-glucoside in wheat and maize

Keywords: LC/MS, cereals, mycotoxins -Fusarium

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Introduction

D3G (structure shown in Figure 1) can be formed by Fusarium infected plants as a response to the production of the mycotoxin DON by the fungi. The existence of a plant conjugate of DON -which might be responsible for partial Fusarium head blight disease of wheat -was proposed in the mid-eighties [START_REF] Miller | Fusarium toxins in field corn. I. Time course of fungal growth and production of deoxynivalenol and other mycotoxins[END_REF]Young et al., 1984). It was assumed that the conjugate might be D3G, which therefore was chemically synthesized [START_REF] Savard | Deoxynivalenol fatty acid and glucoside conjugates[END_REF]. D3G was indeed formed by DON treated maize cell suspension cultures [START_REF] Sewald | Structure elucidation of a plant metabolite of 4-desoxynivalenol[END_REF]. We first detected D3G in naturally contaminated wheat and maize [START_REF] Berthiller | Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry[END_REF]. Recently, D3G was also found in naturally contaminated barley as well as in malt and beer made thereof [START_REF] Lancova | Transfer of Fusarium mycotoxins and 'masked' deoxynivalenol (deoxynivalenol-3-glucoside) from field barley through malt to beer[END_REF].

Glucosylation, a reaction occurring in phase II metabolism of plants, represents a possibility to detoxify xenobiotics (reviewed in [START_REF] Lim | A class of plant glycosyltransferases involved in cellular homeostasis[END_REF]. The detoxification potential of Arabidopsis thaliana [START_REF] Poppenberger | Detoxification of the Fusarium mycotoxin deoxynivalenol by an UDP-glucosyltransferase from Arabidopsis thaliana[END_REF] towards DON has been linked to the UDP-glucosyltransferase UGT73C5, which converts DON to D3G. The importance of this conversion for Fusarium resistance of wheat (Lemmens et al., 2005) has been shown and the quantitative trait locus (QTL) Qfhs.ndsu-3BS has been made responsible for this reaction. However, the fate of D3G after digestion by animals is still largely unknown. Preliminary data from our group indicate that certain intestinal bacteria are able to cleave D3G, converting it back to DON (manuscript in preparation).

Therefore, D3G should be regarded as potentially hazardous for human and animal health. In this work we present occurrence data for D3G along with DON in wheat and maize, obtained by analysis using an LC-MS/MS method (Sulyok et al., 2006). Seventy-seven naturally infected field samples from fields in Central Europe were analyzed for their DON and D3G content to obtain a more comprehensive picture of the co-occurrence of these two compounds.

Materials and Methods

Chemicals

D3G was isolated from DON treated wheat ears [START_REF] Berthiller | Masked mycotoxins: determination of a deoxynivalenol glucoside in artificially and naturally contaminated wheat by liquid chromatography-tandem mass spectrometry[END_REF]. The identity of the substance was confirmed by NMR (data not shown). DON was obtained from Biopure Referenzsubstanzen GmbH (Tulln, Austria) as calibrant in acetonitrile solution.

HPLC grade methanol and acetonitrile were purchased from J.T. Baker (Deventer, The Netherlands). MS grade ammonium acetate and per analysis grade acetic acid were supplied by Sigma-Aldrich (St. Louis, MO, USA). Water for HPLC was purified with a Millipore Milli-Q plus system (Molsheim, France) after reverse osmosis.

Cereal samples

Twenty-three wheat samples from the harvest of 2005 originated from different fields in Austria, Germany and Slovakia. Fifty-four maize samples from the harvest of 2006 originated from different fields in southern Austria. For each cereal, at least 10 incremental samples of 100 g each were taken and combined, according to (EC

Sample preparation

Sample preparation was carried out according to Sulyok et al. (2006) with slight modifications. In brief, 5.00±0.01 g of the ground cereals were extracted with 20 mL acetonitrile/water/acetic acid (79/20/1, v/v/v) for 90 min on a rotary shaker. The filtered raw extracts were diluted 1+1 with acetonitrile/water/acetic acid (20/79/1, v/v/v) and measured with LC-MS/MS without any further clean-up. If an analyte concentration exceeded 1000 ng/mL (above the linear range of the method), the raw extract was diluted 1+9 with the same solvent mixture and the sample was re-analyzed by LC-MS/MS.

LC-MS/MS instrumentation and methodology

An 1100 Series HPLC System (Agilent, Waldbronn, Germany) in conjunction with a QTrap 4000 LC-MS/MS System (Applied Biosystems, Foster City, USA) equipped with a TurboV-ESI source were used for analysis. A multianalyte method was used for analysis in the negative ion mode (Sulyok et al., 2006). A methanol/acetic acid (1%)/ aqueous ammonium acetate (5 mM) linear gradient (from 10 to 97% methanol) was applied for separation on a Phenomenex Gemini-C18 column (4.6 x 150 mm, 5µm). The In order to evaluate the apparent recoveries of DON and D3G, the toxins were spiked separately to ground blank wheat and maize at concentrations of 50 ng/g, 100 ng/g, 200 ng/g, 400 ng/g and 800 ng/g (n=3 each). After extraction and 1+1 dilution, the samples were measured as described. Recovery and repeatability (RSD) were calculated from linear regression curves using Validata (Wegscheider et al., 1999). Limits of detection (LOD) and quantification (LOQ) were calculated, based on signal to noise ratios of 3/1 and 10/1, respectively.

Results and discussion

Due to a slightly modified sample preparation procedure (larger sample weight and a lower dilution factor for the maize samples) to the original method, the recoveries of DON and D3G in both matrices were determined again. Total (apparent) recoveries of 95% for DON and 71% for D3G were obtained for wheat. These values are in complete conformity with the values from the original method (Sulyok et al., 2006). For maize, recoveries of 106% for DON and 130% for D3G were evaluated. These values are also in good agreement to the original method (with 1+1 diluted raw maize extracts). Interestingly, the higher apparent recovery for D3G in maize compared to wheat is caused by a strong matrix enhancement effect in maize, while the extraction recovery is similar for both cereals (Sulyok et al., 2006). For both matrices and both substances the recoveries were constant across the entire range of concentrations. All concentrations for DON and D3G in cereals have been corrected by the obtained recovery factors.

Despite similar retention times (around 7.6 min) for DON and D3G, no cross-talk was obtained even at high concentrations, confirming the selectivity of the used method.

LOQs of about 10 ng/g have been achieved for D3G in both matrices. For DON, the LOQ in wheat was 20 ng/g, in maize 40 ng/g. Relative standard deviations (n=15 for each matrix) were around or below 10% (Table 1).

Twenty-three naturally contaminated wheat samples were analyzed for their concentration of DON and D3G as described above. The results are shown in Table 2, sorted by increasing DON concentrations. Minimum, maximum and average (mean) concentrations are shown in bold letters in the table. DON was found in all measured wheat samples, ranging from 203 to 4130 ng/g. D3G was also detected in all 23 wheat samples at concentrations between 76 and 1070 ng/g. As the molecular mass of D3G (458 g/mol) is higher than the molecular mass of DON (296 g/mol), the D3G/DON ratios are given in mol% in Table 2. The concentrations of D3G in individual wheat samples ranged from 7 mol% to 29 mol% relative to their DON concentrations. In average, 17±5 mol% of the DON concentration in wheat was found as D3G. Fifty-four naturally contaminated maize samples from fields in the southern part of Austria (Styria) were tested for DON and D3G. Again, DON was found to be present in all samples, with concentrations ranging from 42 to 3680 ng/g. D3G also was detected in all maize samples at concentrations between 10 and 763 ng/g. The concentrations of D3G in individual maize samples ranged from 5 mol% to 46 mol% relative to their DON concentrations. On average, 14±8 mol% of the DON concentration was found as D3G in maize.

Due to the use of a multi-analyte method, other mycotoxins have been detected as well, but were not explicitly quantified. Nonetheless, 43 maize samples (80%) contained 15acetyl-DON, 37 maize samples (69%) contained zearalenone, 19 maize samples (35%) contained nivalenol, 18 maize samples (33%) contained moniliformin and 6 maize samples (11%) contained 3-acetyl-DON above their respective detection limits. Similarly, 16 wheat samples (70%) contained zearalenone, 15 wheat samples (65%) contained 15acetyl-DON, 13 wheat samples (56%) contained moniliformin, 7 wheat samples (30%) contained nivalenol, 7 wheat samples (30%) contained 3-acetyl-DON.

Conclusion

Besides the parent mycotoxins, their plant metabolites might also be hazardous for animals and men. Such conjugated or masked mycotoxins can also be easily analyzed with LC-MS methods if appropriate standards are available. To stimulate research efforts in the area of masked mycotoxins, D3G has been made available to the scientific community by the authors. The consistent occurrence of D3G in all tested wheat and the findings of the present study, consumers might currently be exposed to an additional DON load of up to +50% in certain cases arising solely from D3G (assuming complete hydrolysis of D3G to DON during digestion). DON enhances the virulence of Fusarium graminearum on wheat, while it seemingly has little effect on virulence on barley or maize [START_REF] Jansen | Infection patterns in barley and wheat spikes inoculated with wild-type and trichodiene synthase gene disrupted Fusarium graminearum[END_REF]. The increased use of wheat cultivars which carry a major QTL for increased Fusarium resistance and increased ability to conjugate DON [START_REF] Lemmens | The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major QTL for Fusarium head blight resistance in wheat[END_REF] may lead to a higher D3G/DON ratio in the future. In this case the contribution of D3G to overall DON toxicity may no longer be negligible. We therefore conclude that in the future, D3G should be monitored in cereals along with DON, which currently is the only regulated trichothecene in Europe (EC 1126(EC /2007)).

Figure Captions

  ). All samples were ground to a fine powder and stored in paper bags at room temperature until analysis.

[

  M+CH 3 COO] -acetate adducts of the analytes (m/z 355.1 for DON, m/z 517.3 for D3G) were taken as precursor masses. Declustering potentials of -40V for DON and -50V for D3G were chosen. Three product ions each were selected in the third quadrupole (DON: m/z 59.1 (collision energy, CE=-40eV), m/z 295.1 (CE=-16eV), m/z 265.0 (CE=-16eV); /z 427.1 (CE=-30eV), m/z 59.1 (CE=-85eV), m/z 457.1 (CE=-20eV)). The most abundant transitions were used for quantification, the other two as qualifiers. Standardswere diluted separately from their stock solutions with pure water to final concentrations of[START_REF] Lemmens | The ability to detoxify the mycotoxin deoxynivalenol colocalizes with a major QTL for Fusarium head blight resistance in wheat[END_REF] 30, 50, 100, 300, 500 and 1000 ng/mL. After external calibration, 1/x weighted linear calibration functions were generated with Analyst version 1.4.1 (Applied Biosystems).

  the presence of conjugated forms of mycotoxins along with their mycotoxin precursors in cereals and the underestimation of the total DON load. Based on

  Berthiller F, Krska R, Schuhmacher R. 2006. Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rap Commun Mass Spectrom. 20:2649-2659. Wegscheider W, Rohrer C, Neuböck R. 1999. Validata (MS Excel macro for method validation). Version 3.02. Young CJ, Fulcher GR, Hayhoe JH, Scott PM, Dexter JE. 1984. Effect of milling and baking on deoxynivalenol (vomitoxin) content of eastern Canadian wheats. J Agric Food Chem. 32:659-664.

Figure 1 :

 1 Figure 1: Structure of DON-3-β-D-glucoside

Table 1

 1

	:	Method performance
	Table 2:	Concentrations of DON and DON-3-β-D-glucoside in naturally
		contaminated cereals sorted by increasing DON concentration.
		Extreme and average values are shown bold.

http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

Food Additives and Contaminants

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Acknowledgements

The authors thank the Government of Lower Austria, the Christian Doppler Society and the Austrian Science Fund (FWF) for financial support. We also thank Dr. Karl Mayer of the Styrian Chamber of Agriculture for the provision of the maize samples.


Amedeo Pietri
email: amedeo.pietri@unicatt.it

Marco Zanetti

Terenzio Bertuzzi

Distribution of aflatoxins and fumonisins in dry-milled maize fractions

Keywords: Aflatoxins, Fumonisins Food Types: Cereals, Processed foods aflatoxins, fumonisins, maize, dry-milling process, milling fractions

come

Introduction

Maize is one of the most important agricultural crops in the world, both for its potential yield per hectare and its nutritional value. In Italy, the area dedicated to maize cultivation is about 1.1 million hectares, located mainly in Northern Italy, while maize production is about 9.7 million tonnes (Istat, 2006). Although grown principally for animal feed, about 4% of Italian maize production is destined for the milling industry and is processed into products for human consumption.

The natural occurrence in maize of mycotoxins such as aflatoxins, fumonisins, trichothecenes and zearalenone, has been reported in various studies (Vargas et al. 2001;[START_REF] Adejumo | Survey of maize from south-western Nigeria for zearalenone, α and β zearalenols, fumonisin B 1 and enniatins produced by Fusarium species[END_REF][START_REF] Abbas | Aflatoxin and fumonisin contamination of commercial corn (Zea mays) hybrids in Mississippi[END_REF][START_REF] Ali | Natural co-occurrence of aflatoxins and Fusarium mycotoxins (fumonisins, deoxynivalenol, nivalenol and zearalenone) in corn from Indonesia[END_REF][START_REF] Domijan | Fumonisin B1, fumonisin B2, zearalenone and ochratoxin A contamination of maize in Croatia[END_REF]Gonzalez et al. 1999). As regards maize produced in Italy, mycotoxins that deserve special attention are: aflatoxins for their extreme acute and chronic toxicity and because a toxic metabolite (aflatoxin M 1 ) is excreted into the milk of cows fed contaminated feeds; fumonisins for their toxicity and high and widespread contamination that cause several disease states in animals and could affect human health (Pietri et al. 2004;Logrieco et al. 2002;[START_REF] Battilani | Monitoraggio della contaminazione da micotossine in mais[END_REF]. Aflatoxins B 1 , B 2 , G 1 and G 2 are mainly produced by two fungal species: Aspergillus flavus and A. parasiticus, mould species that grow in warm conditions in tropical and sub-tropical regions of the world. Aflatoxin B 1 , (AFB 1 ) the most toxic aflatoxin, is a potent genotoxic carcinogen in laboratory animals and there is strong evidence that it is a liver carcinogen in humans. Fumonisins B 1 , B 2 and B 3 are a group of closely related compounds mainly produced by Fusarium verticillioides and F. proliferatum, fungi that commonly contaminate maize in many growing regions of the world; studies demonstrate the importance of insect damage, drought and temperature stress on fumonisin synthesis. Fumonisins have been shown to be hepatocarcinogenic in male rats and female mice and nephrocarcinogenic in male rats (Voss et al. 2001). Fumonisins have also been associated with the high incidence of human oesophageal cancer in some regions of South Africa and China (Sydenham et al. 1990;Sun et al. 2007). Recent findings suggest that they might increase the risk of neural tube defects in populations consuming large amounts of fumonisin-contaminated maize (Missmer et al. 2006).

Aflatoxin B 1 and Fusarium verticillioides toxins have been categorised by the International Agency for Research on Cancer (IARC) as class 1 (human carcinogen) and 2B (possible human carcinogen), respectively (IARC 1993). In a later evaluation in 2002, the IARC declared fumonisin B 1 (FB 1 ) to be a group 2B carcinogen (IARC 2002).

The EU legislation fixed maximum admissible levels for AFB 1 , total aflatoxins (sum of AFB 1 , AFB 2 , AFG 1 and AFG 2 ) and fumonisins (sum of FB 1 and FB 2 ) in cereals and derived products for direct human consumption (Commission of the European 2006a, 2007). Current limits relevant to maize are given in Table 1.

Communities

Current legislation being very detailed, for maize millers it is vital to know the extent of decontamination that can be obtained by the first step (cleaning) of the process and how these mycotoxins are distributed in final products. Physical procedures generate little heat other than that caused by the operation of the machinery for cleaning, de-husking and abrasion so no significant thermal breakdown of mycotoxins would be expected at this stage. However, moulds and mycotoxins are often concentrated in dust and broken grains or in the outer seed coat of grains. The removal of this material can thus result in a considerable reduction of the mean mycotoxin concentration.

Fumonisin distribution was studied on a laboratory-scale processing apparatus (Katta et al. 1997) or sampling, over 9 months, maize products in a commercial dry-mill [START_REF] Broggi | Distribution of fumonisins in dry-milled corn fractions in Argentina[END_REF]. [START_REF] Brera | Effect of industrial processing on the distribution of fumonisin B 1 in dry-milling corn fractions[END_REF][START_REF] Brera | Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions[END_REF] carried out a study, in a commercial dry-milling plant, of FB 1 , aflatoxins and zearalenone distribution in milled corn fractions from two maize lots showing medium-low contamination levels. These studies indicated that contamination increased, with respect to unprocessed maize, in products like bran, germ and flour of low particle size (animal feed flour), while it decreased in grits and flour of higher particle size, destined for human consumption. No data are available on the effect of the cleaning step.

The aim of our work was to investigate in an industrial dry-mill: (a) aflatoxin and fumonisin distribution in milling products deriving from the processing of two maize lots, that showed (with respect to the Italian situation) medium levels of aflatoxins (AFB 1 , AFB 2 , AFG 1 , AFG 2 ) and fumonisins (FB 1 and FB 2 ) in the first and high levels in the second (about 5 and 8000 µg kg -1 for the first lot, 120 and 13000 µg kg -1 for the second, respectively); (b) the efficacy of maize cleaning steps, carried out at the beginning of the process, on aflatoxin and fumonisin contamination levels. Further, a [insert Table 1 about here]

Materials and methods

Dry-Milling Process

The study was carried out in an industrial dry-milling plant able to process 10 tonnes per hour of maize grain and performed de-germination under partially wet conditions by a conical de-germinator. First, the unprocessed maize was cleaned through three different steps (Figure 1), which included a separator with aspirator (winnower), a dry de-stoner and an intensive scourer coupled with an aspirator. The cleaned maize was soaked in warm water (70°C) for 10 min, to increase moisture to 22%. In these conditions, the germ becomes soft and elastic; also, partial detachment of bran occurs.

After soaking, the maize was decorticated and de-germinated in the conical degerminator, then sifted through a 5-mm sieve. The fraction (large size grits and germ) retained on the sieve was directly conveyed to the plan sifter, while the sieved fraction (flours, bran, small size grits and fractions of germ) was dried to 14% moisture content and conveyed to a turbo-sifter, where the flour of a particle size <2 mm (fine flour), was separated and destined to animal feed. The fraction with a particle size >2 mm was conveyed to a winnower, which aspirated the bran (lighter) sending it to animal feed, while fractions of germ and small size grits were sent to the plan sifter, together with large size grits and germ of particle size >5 mm. The plan sifter separated the products according to their size: those of size >5.5 mm, were conveyed back to the de- germinator, while the smaller ones were separated through a 4 mm sieve. The fractions which were obtained were sent to different gravity tables, to separate germ from small and large size grits. Fine grits were processed again using a horizontal roller mill and transformed into flour, that was conveyed to a plan sifter and divided between coarse flour (300-850 µm), fine flour (<300 µm) and residual germ (about 1%). Undersized flour, bran and cleaning waste, were processed with a hammer mill for the production of animal feed flour.

[insert Figure 1 about here]

Samples

Two maize lots (maize 1 and 2), with different aflatoxin and fumonisin contamination, were processed and sampled. Both were FAO 600 class hybrids, with flinty endosperm. Samples of unprocessed maize and derived fractions were drawn from twelve opening slits of the plant (numbered in Figure 1). In order to obtain a dynamic representative sampling (corresponding to the flow of different products), a primary sample of about 300 g was collected from each slit every 90 seconds for one hour (40 samples), according to the Regulation 2006/401/EC (Commission of the European Communities 2006b). From the cleaning plant were collected: unprocessed maize (1), waste of the winnower (2), of the dust filter (3), of the intensive scourer (4) and cleaned maize (5).

From the de-germination process were collected: bran (6), germ (7), coarse grits (8), fine grits (9) and animal feed flour (12). From the refining step were collected: coarse (10) and fine flour (11). For each collecting-slit, the 40 primary samples were mixed, ground with a hammer mill (1 mm sieve), and homogenized for 10 min using a

Analytical Standards

Aflatoxin and fumonisin standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). For each aflatoxin, a stock solution of 5-8 µg ml -1 was prepared in benzene:acetonitrile (98:2, v/v, 2 ml) and stored at -20°C. The solution was calibrated spectrophotometrically at 350 nm (AOAC International, 2005). The working standard solution was prepared after evaporation under nitrogen of an aliquot (100 µl) of the stock solution and re-dissolution in chloroform (10 ml) by ultrasonication. An aliquot (100 µl) of this solution was evaporated under nitrogen and re-dissolved in the HPLC mobile phase (0.5-5 ml), to obtain calibrant solutions at individual concentrations of AFB 1 , AFB 2 , AFG 1 , AFG 2 between 0.3 and 6 µg l -1 . FB 1 and FB 2 (1 mg) were separately dissolved in 10 ml acetonitrile:water (1:1, v/v); the concentration was calculated using the weight indicated by the manufacturer. These solutions were diluted to obtain HPLC calibrant solutions in acetonitrile:water (30:70, v/v, acidified with 0.4% acetic acid) at individual concentrations of FB 1 and FB 2 between 2.5 and 50 µg l -1 .

Analysis for aflatoxins and fumonisins

Aflatoxins were analysed according to the method of Stroka et al. (1999). Aflatoxins were extracted from 25 g of sample with 250 ml methanol:water (80:20, v/v), using a rotary-shaking stirrer for 45 min. After filtration through a folded filter-paper, an aliquot of the filtrate (5 ml) was diluted with distilled water (45 ml) and the solution was purified through an immunoaffinity column (R-Biopharm Rhône LTD, Glasgow, Scotland, UK). After washing of the column with 5 ml distilled water, aflatoxins were eluted into a graduated glass vial with methanol (2.5 ml). The eluate, concentrated to 1.0 ml under a gentle stream of nitrogen, was brought to 2 ml with acetonitrile:water (25:75, v/v), and vortex-mixed for a few seconds; then, the extract was filtered (Millipore Corporation, Bedford, Massachussetts, USA, HV 0.45 µm) and injected (30 µl). Analysis was performed using an HPLC instrument, consisting of two PU-1580 chromatographic pumps, an AS 1555 sampling system, a FP 1520 fluorescence detector and a post-column derivatization system (Jasco Corporation, Tokyo, Japan); the instrument was controlled by Borwin 1.5 software (Jasco). A Superspher RP-18 column (4 µm particle size, 125x4 mm i.d., Merck) was used at ambient temperature, with a mobile phase of water:methanol:acetonitrile (64:23:13, v/v/v), at 1.0 ml min -1 . A solution of pyridinium bromide perbromide (25 mg in 500 ml of HPLC-grade water) was used as a derivatizing agent. The flow of the post-column derivatizing solution was set at 0.1 ml min -1 and the reaction tubing was 500 µl. The detector was set at λ ex =365 nm and λ em =440 nm. FB 1 and FB 2 content was analysed according to the method proposed by Visconti et al. (2001). Fumonisins were extracted from 10 g of sample in a plastic centrifuge bottle with 50 ml of acetonitrile:methanol:water (25:25:50, v/v/v). After extraction for 45 min using a rotary-shaking stirrer and centrifugation at 4500 g for 6 min, the supernatant was poured into a flask; another 50 ml of the same solution was added to the residue in the centrifuge bottle, and a second extraction performed for 30 min. The combined extracts were filtered through a folded filter-paper. An aliquot of 2 ml was diluted with 20 ml of 0.1 M phosphate buffered saline (PBS, pH=7.4) and purified through an immunoaffinity column (R-Biopharm Rhône Ltd, Glasgow, Scotland, UK); after washing the column with PBS (2 ml), the fumonisins were slowly eluted (0.5 ml min -1 ) with methanol (6 ml) into a graduated glass vial; subsequently, the eluate was concentrated to 2 ml under a gentle stream of nitrogen. Analysis was carried out using a LC-MS/MS system, consisting of a LC 1.4 Surveyor pump (Thermo-Fisher Scientific, San Jose, CA, USA), a PAL 1.3.1 sampling system (CTC Analitycs AG, Zwingen, Switzerland) and a Quantum Discovery Max triple-quadrupole mass spectrometer; the system was controlled by Excalibur 1.4 software (Thermo-Fisher). After dilution of the extract (0.1 ml brought to 1 ml) with acetonitrile:water (30:70 v/v, acidified with 0.4% acetic acid), the fumonisins were separated on a Betasil RP-18 column (5 µm particle size, 150 x 2.1 mm, Thermo-Fisher) with mobile-phase gradient acetonitrile-water (both acidified with 0.4% acetic acid) from 25:75 to 55:45 in 9 min, then isocratic for 3 min; the flow rate was 0.2 ml min -1 . Ionisation was carried out with an ESI interface (Thermo-Fisher) in positive mode as follows: spray capillary voltage 4.0 kV, sheath and auxiliary gas 35 and 14 psi, respectively, temperature of the heated capillary 270 °C.

The mass spectrometric analysis was operated in selected reaction monitoring (SRM).

For fragmentation of [M+H] + ions (722 m/z for FB 1 , 706 m/z for FB 2 ), the argon collision pressure was set to 1.5 mTorr and the collision energy to 36 V. The selected fragment ions were: 704, 352 and 334 m/z for FB 1 , 688, 336 and 318 m/z for FB 2 .

Quantitative determination was performed using LC-Quan 2.0 software (Thermo-Fisher).

Results and discussion

Yields of the milling-corn fractions

Yields of the different fractions, calculated with an approximation of 1%, are reported in Table 2. The two maize lots had different characteristics, maize 2 being visually dustier and with a higher amount of broken kernels with respect to maize 1; as a consequence, the cleaning waste was 6 vs. 3% of the processed product for maize 2 and 1, respectively. Further, maize 2 yielded a lower amount of undersized flour deriving from the turbo sifter, with respect to maize 1 (17 vs. 20%). As a consequence, the yield of animal feed flour was the same for maize 1 and 2 (28%). For both maize lots, the ratio between coarse and fine flour, after refining of fine grits, was 85:15. and 1000 µg kg -1 for aflatoxins and fumonisins, respectively. Average recovery values were: 97.8∀1.6% for AFB 1 and over 93% for the other aflatoxins, 95.5∀1.9% for FB 1 and 93.6∀2.1% for FB 2 . A certified reference material (ground corn, R-Biopharm Rhône LTD), with an AFB 1 contamination of 4.1∀1.0 µg kg -1 , was also analysed; the result was 4.2∀0.2 µg kg -1 . The results of the analyses were not corrected for recovery.

[insert

The limit of detection (LOD, signal-to-noise ratio of 3:1) and of quantification (LOQ, signal-to-noise ratio of 10:1) were respectively 0.02 and 0.05 µg kg -1 for aflatoxins, 10 and 30 µg kg -1 for fumonisins.

Aflatoxin distribution

AFB 1 and AFG 1 were detected in both maize lots, but AFB 2 and AFG 2 only in maize 2 (Table 3). The AFB 1 level of maize 1 (3.6 µg kg -1 ) was lower than the value fixed by EC Regulation 1881/2006 for unprocessed maize (5 µg kg -1 ); however, considering the final products, only coarse flour was within the limit for human consumption (2 µg kg -1 ), while coarse and fine grits slightly exceeded this limit. After the cleaning steps, maize 1 showed an AFB 1 concentration slightly lower than the unprocessed one (3.3 against 3.6 µg kg -1 , -8%). Bran and germ deriving from de-cortication and degermination steps, resulted as more contaminated than cleaned maize (+127 and +197%, respectively). Animal feed flour, that included cleaning waste, bran, and undersized flour ground together, showed an AFB 1 level close to raw maize. The results obtained for maize 1 basically accord with those reported by [START_REF] Brera | Effect of industrial processing on the distribution of aflatoxins and zearalenone in corn-milling fractions[END_REF]: processing two maize lots with an AFB 1 concentration below 3 µg kg -1 , they observed an increase of contamination in bran, germ and animal feed flour and a consequent decrease in grits and flour destined for human consumption, that showed values below 2 µg kg -1 . Maize 2 was much more contaminated (91.1 µg kg -1 ); after cleaning it showed an AFB 1 level decidedly lower (39.5 µg kg -1 ) with respect to the unprocessed one (-57%), as cleaning steps removed the most contaminated fractions (broken kernels, dust, etc.).

Bran and germ of maize 2 showed much higher AFB 1 concentrations (+379 and +629%, respectively) compared to cleaned maize. As a consequence, unlike maize 1, the contamination level decreased remarkably in grits (-80%) and increased (+141% with respect to raw maize) in animal feed flour; however, the latter could not be used, as the value (0.219 mg kg -1 ) exceeded the limit of 0.02 mg kg -1 in force in the EU (Commission of the European Communities 2002). None of the milling-corn fractions destined for human consumption showed a concentration below the legal limit (2 µg kg -1 ), but they could be used as feeds. Considering the AFB 1 percent distribution (Table 3), it is evident that cleaning steps led to very different results for the two maize lots, removing 11 vs. 59% of the toxin from maize 1 and 2, respectively. Analogously, very different results were observed in de-cortication and de-germination steps; toxin removal was 30 vs. 72% for cleaned maize 1 and 2, respectively. Consequently, the AFB 1 percentage remaining in coarse and fine grits, was 55 vs. 13%, for cleaned maize 1 and 2, respectively. Fine grits were subsequently processed to obtain coarse and fine flour, in the ratio 85:15; for maize 1, 61% of the AFB 1 amount in fine grits (7.2% of the total) was present in coarse flour and 16.1% in fine flour; for maize 2, the corresponding percentages were 64.3 and 9.0%. The lower figure was due to a small amount of remaining germ fraction (about 1% of unprocessed maize), more contaminated, that was removed in this step of the process. The different AFB 1 percentage (26.4% against 67.5% for maize 1 and 2, respectively) collected in animal feed flour, was mainly due to the different contribution made by the waste of the A similar trend was observed for the other aflatoxins.

[insert Table 3 about here]

Fumonisin distribution

Both maize lots turned out to be contaminated with FB 1 and FB 2 (Table 4) at levels exceeding the limit (4000 µg kg -1 ) in force in the EU. Maize grains obtained after cleaning steps showed fumonisin concentrations lower (especially maize 2) than the unprocessed ones (-11 and -34% for FB 1 ). Bran showed higher contamination levels with respect to cleaned maize (+50 and +167% for FB 1 ); germ turned out to be less contaminated with respect to bran in both maize lots. Coarse and fine grits, fractions intended for human consumption, showed much lower FB 1 concentrations when compared to cleaned maize (-90 and -88% for maize 1 and -90 and -73% for maize 2).

Besides, fine grits and fine flours, showed a level higher than the coarse ones.

Therefore, as already found by [START_REF] Brera | Effect of industrial processing on the distribution of fumonisin B 1 in dry-milling corn fractions[END_REF], contamination increases in lower particle size final products. Coarse grits for both maize lots, fine grits and coarse flour for maize 1, showed a contamination level (FB 1 + FB 2 ) below 1400 µg kg -1 , which is the limit fixed by the EU. Animal feed flour, which collected bran and cleaning waste, was three times more contaminated than unprocessed maize. Contamination levels found in different corn-milling fractions were similar to those found in previous studies (Katta et al., Broggi et al., Brera et al.), in which there was always reported an increase of concentration in bran, germ and animal feed flour and a decrease in products intended for human consumption, like grits and flours. As regards the FB 1 percentage distribution, the cleaning plant removed 14% and 38% of the toxin for maize 1 and 2, respectively; regarding cleaned maize, de-cortication and de-germination processes removed altogether 12% and 27% of the toxin, while 6.8% and 8.8% remained in coarse and fine grits. It is evident that the highest fraction of FB 1 remained in the undersized flour separated from the turbo sifter and conveyed into animal feed flour; this product gathers 83.3 and 90.9% of the total FB 1 present in uncleaned maize 1 and 2, respectively. A similar trend has been observed for FB 2 .

[insert Table 4 about here]

Mass balance

Mass balances of aflatoxins and fumonisins for the cleaning step, for the degermination process and for the complete process are reported in Table 5. The mass balance of the complete process for AFG 1 in maize 1 was relatively low (79.3%), probably because of a high analytical error associated with the low level of the mycotoxin; except for this result, mass balances of mycotoxins in both processes were in the range 90-110%.

Thus, it can be concluded that the dynamic sampling was effective and the results were altogether accurate.

[insert Table 5 about here]

Conclusions

The extent of decontamination obtained from the cleaning step was decidedly higher for a "dirty" maize (maize 2) with respect to a "normal" maize (maize 1). In this step, the percent toxin reduction in maize 2, if calculated in terms of both concentration and mass, was 57-59% and 34-38% for AFB 1 and FB 1 , respectively. For maize 1, the corresponding values were 8-11% and 11-14% for AFB 1 and FB 1 , respectively.

The extent of decontamination obtained from the cleaning step of maize 2, was higher for aflatoxins than for fumonisins. Probably, during harvesting, handling and transportation, a fraction of the aflatoxin contaminated kernels broke and produced powder; therefore their removal contributed significantly to decontamination. For maize 2, discarding of the cleaning waste would decrease the yield of animal feed flour from 28% to 22%, but the total amount of AFB 1 and FB 1 residual in the product would decrease from 67.5% to 8.2% and from 90.9% to 53.2%, respectively. Generally, it is evident that an alternative use of cleaning waste would substantially improve the quality of animal feed flour.

As regards products after the cleaning step, a different distribution of the two toxins was observed. There was a large amount of AFB 1 in the germ fraction, of 18.5% and 46.7% of the total for maize 1 and 2, respectively; for bran the corresponding values were 11.8% and 25.6%; most of the remaining AFB 1 was in coarse grits. As regards FB 1 , 4.3% and 12.7% occurred in germ, 7.7% and 14.1% in bran, for maize 1 and 2, respectively; the amount remaining in coarse grits was low (5.5-5.6%), while most was collected in undersized flour (81-64%), that was intended for animal feed. It is evident that aflatoxin contamination of maize kernels was more superficial and concerned mainly the germ, while fumonisin contamination affected the inner layers of kernels. Gonzalez, H., Martinez, E., Pacin, A., Resnik, S., Sydenham, E. 1999 Logrieco, A., Mulè, G., Moretti, A., Bottalico, A. 2002. Toxigenic Sun, G., Wang, S., Hu, X., Su, J., Huang, T., Yu, J., Tang, L., Gao, W., Wang, J. 2007.

  the mycotoxins was carried out, in order to verify the accuracy of the results.

  For each product, two sub-samples of 1.5 kg were withdrawn and stored at -20°C until the time of analysis. The percentage yields of the different fractions were calculated taking into account the technical specifications of the plant and from measurements carried out by plant-technicians during processing of the two maize lots, before sampling.

  Both for aflatoxins and fumonisins, four replicates were analysed for each sample.Mass balanceBoth for aflatoxins and fumonisins, mass balances of the cleaning step [Σamount (cleaning waste+cleaned maize) /amount (unprocessed maize) ]*100, of the de-germination process [Σamount (animal feed flour-cleaning waste+grits+germ) /amount (cleaned maize) ]*100 and of the total milling process [Σamount (animal feed flour+coarse grits+flours+germ) /amount (unprocessed maize) ]*100, were calculated.

  Fusarium species and mycotoxins associated with maize ear rot in Europe. Mycotoxins in Plant Disease. European Journal of Plant Pathology, edited by A. Logrieco, J.A. Bailey, L. Corazza & B.M. Cooke (Dordrecht:Kluver), 597-609. Missmer, S.A., Suarez, L., Felkner, M., Wang, E., Merrill, A.H., Rothman, K.J., Hendricks, K.A. 2006. Exposure to fumonisins and the occurrence of neural tube defects along the Texas-Mexico border. Environmental Health Perspectives 114(2): 237-241.

  .,Bertuzzi, T., Pallaroni, L., Piva, G. 2004. Occurrence of mycotoxins and ergosterol in maize harvested over 5 years in Northern Italy. Food Additives and Contaminants 21 (5):479-487. Stroka, J., Petz, M., Joerissen, U., Anklam, E. 1999. Investigation of various extractants for the analysis of aflatoxin B 1 in different food and feed matrices. Food Additives and Contaminants 16 (8): 331-338.

Fumonisin B 1

 1 contamination of home-grown corn in high risk areas for esophageal and liver cancer in China. Food Additives and Contaminants 24 (2): 181-185. Sydenham, E.W., Thiel, P.G., Marasas, W.F.O., Shephard, G.S., Van Schalkwyk, D.J., Koch, K.R. 1990. Natural occurrence of some Fusarium mycotoxin in corn from low and high esophageal cancer prevalence areas of the Transkei, Southern Africa. Journal Agriculture Food Chemistry 38: 1900-1903. Vargas, E., Preis, R., Castro, L., Silva, C. 2001. Co-occurrence of aflatoxins B 1 , B 2 , G 1 , G 2 , zearalenone and fumonisin B 1 in Brazilian corn. 2001. Food Additives and Contaminants 18(11): 981-986. Visconti, A., Solfrizzo, M., Girolamo, A. de. 2001. Determination of fumonisins B 1 and B 2 in corn and corn flakes by liquid chromatography with immunoaffinity column cleanup: collaborative study. Journal of AOAC International 84 (6): 1828-1837. Voss, K., Riley, R., Norred, W.P., Bacon, C.W., Meredith, F., Howard, P., Plattner, R., Collins, T., Hansen, D., Porter, J. 2001. An overview of rodent toxicities: liver an kidney effects of fumonisins and Fusarium moniliforme. Environmental Health Perspectives 109, suppl.2: 259-266.

Figure 1 .

 1 Figure 1. Layout of the industrial plant showing sampling points (numbered) and final products (bold type). 254x190mm (72 x 72 DPI)

  Table 2 about here]

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	Performance of the methods
	Recovery percentages were evaluated by spiking known blank samples at levels of 2

  . Natural cooccurrence of fumonisins, deoxynivalenol, zearalenone and aflatoxins in field trial corn in Argentina. Food Additives and Contaminants 16(12): 565-569.

	International Agency for Research on Cancer (IARC). 1993. Some naturally occurring
	substances, food items and constituents, heterocyclic aromatic amines and
	mycotoxins. IARC Monographs on the Evaluation of Carcinogenic Risk to
	Humans No. 56. Lyon: IARC: aflatoxins p.245; Fusarium verticillioides toxins
	p.445.
	International Agency for Research on Cancer (IARC). 2002. Summaries & Evaluations
	No. 82. Lyon: IARC: fumonisin B 1 p.301.
	Istat 2006.Available:www.istat.it/agricoltura/datiagri/coltivazioni/anno2006/corg2006.
	Accessed 02 October 2007.
	Katta, S.K., Cagampang, A.E., Jackson, L.S., Bullerman, L.B. 1997. Distribution of
	Fusarium molds and fumonisins in dry-milled corn fractions. Cereal Chemistry 74
	(6): 858-863.

Table 1 .

 1 EU maximum admissible limits for AFB 1 , total aflatoxins (sum of AFB 1 , AFB 2 , AFG 1 , AFG 2 ) and fumonisins (sum of FB 1 and FB 2 ) in maize and derived

	products.
		AFB 1	Sum of AFB 1 , AFB 2 , AFG 1 and AFG 2	Sum of FB 1 and FB 2
		(µg kg -1 )	(µg kg -1 )	(µg kg -1 )
	Unprocessed maize	5.0	10.0	4000
	Maize for direct human consumption	2.0	4.0	1000
	Maize-based breakfast	-	-	800
	Milling fractions of maize with particle size > 500 micron	-	-	1400
	Milling fractions of maize with
	particle size # 500 micron	-	-	2000

Table 2 .

 2 Yield of maize milling fractions.

	Maize milling fractions	Yield maize 1 (%) Yield maize 2 (%)
	Unprocessed maize	100	100
	Waste of aspirator	2	3
	Waste of dust filter	0,2	1,5
	Waste of intensive scourer	0,8	1,5
	Cleaned maize	97	94
	Bran	5	5
	Germ	6	6
	Coarse grits	55	55

Table 3 :

 3 Aflatoxin levels (mean of 4 replicates, µg kg -1 ), relative standard deviation (RSD, %) and AFB 1 relative distribution (%) in maize-milling fractions referred to unprocessed maize and (between parentheses) to cleaned maize.

		µg kg -1 RSD %	Distribution %	µg kg -1 RSD %	µg kg -1 RSD %	Distribution %	µg kg -1 RSD %	µg kg -1 RSD %	µg kg -1 RSD %
	Unprocessed maize	3.6	11.1	100	1.7	5.9	91.1	3.8	100	3.4	8.8	26.3	1.5	1.1	9.1
	Waste of aspirator	3.4	11.8	1.9	1.8	11.1	138.3	3.9	4.6	38.1	3.4	26.3	9.5	1.6	12.5
	Waste of dust filter	17	9.4	1.0	8.3	8.4	1296	2.0	21.3	57.8	2.4	290.3	2.3	9.1	5.5
	Waste of intensive scourer	11.5	12.2	2.6	6.1	8.2	1554	0.8	25.6	54.5	2.9	285.5	2.5	9.4	5.3
	Cleaned maize	3.3	15.1	88.7 (100)	1.1	9.1	39.5	6.1	40.7 (100)	1.8	11.1	13.9	4.3	0.7	14.3
	Bran	7.5	9.3	10.5 (11.8)	3.9	7.7	189.4	4.5	10.4 (25.6)	7.3	10.9	42.7	3.5	1.1	9.1
	Germ	9.8	9.2	16.4 (18.5)	5.2	9.6	287.9	1.0	19.0 (46.7)	16.7	3.0	81.9	1.5	5.0	6.0
	Coarse grits	2.7	11.1	41.7 (47.0)	1.1	9.1	7.6	3.9	4.6 (11.3)	0.4	25.0	1.9	5.3	0.3	16.7
	Fine grits
	from which:	2.3	8.7	(8.1) 7.2	1.0	10	7.8	5.1	(2.2) 0.9	0.7	14.3	2.4	8.3	0.6	16.7
	coarse flour	1.7	11.8	61.0	0.8	12.5	5.9	5.1	64.3	0.6	16.7	2.0	10.0	0.5	10.0
	fine flour	2.5	12.0	16.1	1.2	8.3	4.7	6.4	9.0	0.5	20.0	1.4	14.3	0.4	12.5
	Animal feed flour	3.4	11.8	26.4	1.1	9.1	219.6	2.5	67.5	8.8	3.4	48.5	7.4	1.7	11.8

Table 5 :

 5 Aflatoxin and fumonisin mass balance (%) for maize 1 and 2. Figure 1. Layout of the industrial plant showing sampling points (numbered) and final products (bold type).

	Mass balance	AFB 1 AFB 2 AFG 1 AFG 2	FB 1	FB 2
	Cleaning step Maize 1 Maize 2	94.2 96.3	-109.1	93.8 96.2	-96.0	100.4 95.6	104.0 102.2
	De-germination process Maize 1 Maize 2	97.2 99.5	-108.0	110.0 99.5	-99.5	91.2 114.6	89.8 106.1
	Complete milling process Maize 1 Maize 2	90.0 91.7	-110.0	79.3 92.1	-92.1	92.9 104.3	95.0 106.2

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


Gianna Pierlanfranco D'agaro

Molin Dal

Emanuela Zamparo

Tatiana Rossi

Michele Minuzzo

Marina Busetti

Daniela Santon

Cesare Campello

D ' Agaro
email: dagaro@burlo.trieste.it

Pierlanfranco D'

Gianna Dal

Michele Micuzzo

Prof D ' Pierlanfranco

Agaro

Mrs Claudia Biagi

Fabia Petronio

Epidemiological and molecular assessment of a Rubella outbreak in North-Eastern Italy

Keywords: Rubella, outbreak, genotyping, sequences, seroepidemiology Rubella, outbreak, genotyping, seroepidemiology 13 14

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

1 Rubella is a mild exanthematic illness whose public health relevance is due to 2 infection in the first trimester of pregnancy, leading potentially to the congenital 3 rubella syndrome (CRS). CRS comprises a lengthy list of abnormalities, which 4 include commonly deafness, ocular and cardiac defects and mental disability [Miller 5 et al., 1982]. 6 The rubella vaccines are highly efficient for the prevention of rubella infection and Table III shows the results of the seroprevalence survey in women of childbearing 11 age living in Trieste. From 20 to 44 years of age, the prevalence of rubella antibody 12 was distributed evenly in all age groups (range 92.9-95.3% of seropositivity). The 13 overall prevalence rate in 1416 women of childbearing age was 94.5%. the required rate of immunity to rubella [START_REF] Nardone | Comparison of 31 rubella seroepidemiology in 17 countries: progress towards international disease control 32 targets[END_REF]. In Italy, a cross-2 sectional nation-wide survey was carried out in 2004 on sera from subjects of all age 3 groups, both male or female [START_REF] Rota | Rubella seroprofile of the Italian population: an 8-year comparison[END_REF]. The most significant results were the 4 proportion of seronegative males higher than that of female. This is probably due to 5 selective vaccination of pre-pubertal girls and adult women which was undertaken 6 widely, if irregularly, in the past and is in line with the finding of a higher morbidity 7 in males during the outbreak described above. With regards to immunity in women of 8 childbearing age, the proportion of seropositive women ranged from 92% to 95%,

MATERIALS AND METHODS

7 4 A

 4 CRS worldwide[WHO, 2005a;[START_REF] Pandolfi | Prevention of congenital rubella 36 and congenital varicella in Europe[END_REF]. In Italy, universal vaccination 8 against rubella, measles and mumps has been recommended since the early 1990s for 9 all children at fifteen months of age [Ciofi degli Atti et al., 2004; Bonanni et al., 10 2007; Spiteri et al., 2008; Pandolfi et al., 2009]. Before the 1990s, vaccination was 11 offered actively to pre-pubertal girls with a high variability of compliance. In 12 addition, in 2003, the National Plan for Measles and Congenital Rubella Control 13 launched by the Italian Ministry of Health recommended strongly immunization of 14 seronegative women of childbearing age. The plan introduced also two other 15 strategies, i.e. universal second doses at 5-6 years and a catch-up vaccination of 16 children attending primary school. Despite these efforts, rubella is still endemic in 17 Italy as documented by outbreaks recorded in 1997 and 2002, while the rate of CRS 18 is so far higher than the target established by WHO of less than one case/10 5 live 19 births by 2010. In Italy, data on CRS dates only from 2005 when the CRS, as well as 20 rubella infection during pregnancy, was established as a notifiable syndrome. Thus, 21 between 2005 and 2008, seven confirmed or probable cases of CRS and 31 cases of 22 pregnant women were reported, with a sharp rise during the first 1 months of 2008.It is worth noting that only a number of Italian regional health 2 services report both CRS cases and rubella infection during pregnancy, hence the 3 prevalence of CRS in Italy is underestimated[START_REF] Giambi | Alarm rubella: promote 12 actionsfor capable vaccinate women of childbearing age[END_REF]. number of epidemics were recorded in Italy in 2008, one of which is described 5 below together with the genetic characterisation of rubella virus (RUBV) strains. In 6 addition, since the main strategic solution for prompt control of congenital rubella 7 syndrome is focused on the vaccination of susceptible women of childbearing age, 8 the results of a seroprevalence survey carried out in women in the same geographical 9 area sometime before this outbreak are also reported.

  rubella is a mandatory notifiable disease. Since January, 2008, any notified 16 case with fever, typical rubella-like rash and lymphoadenopathy was registered as a 17 suspected case at the Prevention Department of the Health District ASL 6, which 18 corresponds roughly to the administrative province of Pordenone with an area of 19 about 300,000 inhabitants. The suspected case was interviewed over the telephone or 20 asked to complete a questionnaire on demographic data and history of exanthemata, 21 vaccinations and contacts. Following informed consent, the patients provided clinical 22 samples for laboratory investigation.

23 A 9 10 Diagnostic 11 ( 4 P<0 4 From 2

 2391011442 confirmed case was defined as a patient with documented seroconversion, with or 24 without detection or isolation of virus from clinical samples. No possible case, i.e. a 25 with exposure to rubella but without laboratory evidence, 1 was considered in this survey.2 Between January and October, 2006 a survey on the prevalence of rubella antibody 3 was carried out on women of childbearing age (mostly pregnant women) from Trieste 4 attending the Maternal-Children Hospital for TORCH serology evaluation. Since in 5 this area almost all pregnant women are screened for Toxoplasma gondii, all sera 6 were also tested for rubella antibody, irrespective of a specific request. In this way a 7 representative sample of all women of childbearing age living in a limited urban area 8 was enrolled.Serology serology was carried out with a Rubella IgG and IgM EIA commercial kit Dia-Sorin, Saluggia, Italy). Rubella infection was confirmed when IgM antibody 12 was present unequivocally. In the event of an equivocal result, a second serum 13 sample was required to ascertain seroconversion. For the seroprevalence survey, 14 rubella susceptibility was confirmed when serum had an IgG titre less than 10 15 U.I./mL. 16 Virological and molecular tests 17 Virological and/or molecular tests were carried out on throat swabs, urine and 18 peripheral blood in EDTA. Nucleic acids were extracted with the NucliSens easyMag 19 System (Biomerieux Italia, Firenze, Italy) and rubella RNA detection was carried out 20 with a commercial nested RT-PCR and using biotin-labelled probes on streptavidin-21 coated microplate wells (ProDect Rubella, Bcs Biotech, Cagliari Italy). Pharyngeal 22 were seeded on VERO cells, incubated for 10 days and submitted to 1 one blind passage. Supernatants of cell cultures with or without cytopathic effects 2 were submitted to RNA extraction and subsequent RT-PCR. The set of primers used 3 for virus detection in cell cultures were E1.5 (+), E1.6R (-) for the first round of 4 amplification and E1.7 (+) and E1.8R (-) for the nested PCR (Jin and Thomas, 2007). 5 The sequences of the 281 bp amplicon in the 3' end of the E1 region included 225 bp 6 (9245-9469) in the 739 nt window required for genotyping [WHO, 2005b]. The 7 amplicons were concentrated and desalted using the Microcon 100 device (Amicon, 8 Beverly, MA), and were sequenced by Big dye terminator chemistry, v. 3.1, under 9 standard conditions (Applied Biosystems, Foster City, CA) using E1.7 and E1.8R 10 primers. Reaction products were analysed by the ABI 310 Genetic analyser (Applied 11 Biosystems, Foster City, CA). The assemblage of sequences was carried out by the 12 Sequencer package 4.5 of Gene Codes Corporation (Ann Arbor, MI). Phylogenetic 13 and molecular analyses were carried out using MEGA version 4 [Kumar et al., 2004]. 14 A phylogenetic tree was constructed by the Neighbor-Joining method on amino acid 15 sequences; the Kimura two-parameter method was used to calculate nucleotide 16 substitutions, and a bootstrap of 500 replicates confirmed the significance of tree 17 topology. A search for highly similar sequences was performed with the Megablast 18 Algorithm [Zhang et al., 2000]. Sequences generated in this study were deposited in 19 GenBank under the following accession numbers: HM0511264-HM051279. Rubella 20 reference strains [WHO, 2007] and previously reported sequences of Rubella E1 21 region from GenBank were included in the phylogenetic tree. as frequency, proportion or mean, as appropriate. The Chi square 2and Fisher exact tests were used to test differences in frequencies and the Student-t 3 test to assess differences in mean. The differences were considered significant when cases were notified, all were submitted to laboratory testing and 111 8 cases were confirmed. Figure1shows the distribution by age of notified cases of 9 rubella together with the number and the proportion of laboratory confirmed cases. 10 Only one case of rubella was confirmed in the highly vaccinated cohort of children 11 (<10 years), while 91% of confirmed cases occurred in the older cohorts. It is worth 12 noting that an alternative viral etiology was established in eight patients: three were 13 positive for HHV6, three positive for parvovirus B19 and two were EBV-positive. 14 Thus, only laboratory-confirmed cases have been used to describe the outbreak and to 15 analyse it in relation to time, place and personal features. 16 The distribution of cases by month and origin is shown in Figure 2. The epidemic 17 started in a home for young adults disabled mentally and physically . The first case of 18 rubella was notified on 10 January, 2008. He was a 25 years old unvaccinated male 19 resident in a protected care setting. The exposure of the index case to a rubella source 20 could not be discovered. The outbreak involved another disabled home attended by 21 some patients and health care workers of the first home. Eighteen cases were 22 among subjects attending the care facilities. The mean age was 36.6 years 1and the male/female ratio was 2 (TableI). No one had been vaccinated previously 2 against rubella. The last case of institutional rubella was observed on 23 March, February 2008 a community-acquired outbreak took place involving a total of 5 93 subjects: 69 males with a mean age of 25 years and 24 were females with a mean 6 age of 24.5 years. All but two subjects were unvaccinated. Almost all cases occurred 7 in high schools and in industrial settings. Three pregnant women were infected at 8 different gestational weeks. Two pregnancies were terminated and tissues of one 9 fetus were positive for rubella virus. A clear relationship between the two subsequent 10 waves could not be established on the grounds of epidemiologic assessment. The 11 epidemic ended on 3 June, 2008 following the vaccination of susceptible subjects 12 attending the residences for the disabled (56 subjects) and general control measures 13 launched in the entire region. 14 A phylogenetic tree of the sixteen sequences obtained from the 281 bp amplicons in 15 the E1 region is shown in Figure 3 together with some reference sequences available 16 in Genbank. All viruses were related to the 2B genotype with a highly significant 17 bootstrap value (>70). Only one strain (RVi/ FVG.ITA/06.08/2) proved distinct 18 showing a non-synonymous mutation in nucleotide 2884 of the ORF2 sequence, 19 corresponding to F962G mutation. In addition, the nucleotide sequence analysis 20 revealed two viral populations showing synonymous mutations in positions 2904 and 21 2955 (Table II). During the first epidemic phase, viruses had a Cytosine both in 22 2955, while eight strains isolated from subjects in the general 1 population during the second wave, had Thymine in the same positions. The CC 2 pattern was significantly more frequent in institutionalized patients than in the 3 general population (Fisher exact test: P=0.006) and in the first half of the epidemic 4 than in the second one (Fisher exact test: P=0.005). Interestingly, one sequence (RVi/ 5 FVG.ITA/08.08) had a mixture of the two patterns (YY). The Blast analysis of the 6 280 bp nucleotide sequence with the CC pattern revealed a complete identity with the 7 RVs/London.GRB/9.08 acquired in Italy (GenBank FJ774989) while the TT pattern 8 was very similar to the strain isolated in London in 2006 (RVs/London.GBR/07.06) 9 and in Milan, Italy, in 1994 (RVi 5298-ITA94).

10

 10

10 [

 10 of the largest rubella outbreaks recorded in a highly vaccinated population 17 of a limited area in Italy with about 300,000 inhabitants. It is worth noting that the 18 description of the outbreak is based only on laboratory confirmed cases since the[START_REF] Jin | Application of molecular and serological assays to case based 20 investigations of rubella and congenital rubella syndrome[END_REF] predictive value of the clinical diagnosis of rubella is unreliable, mainly in children 20 with a high immunisation cover. According to findings obtained in similar highly[START_REF] Kumar | MEGA3: Integrated software for Molecular Evolutionary 22 Genetics Analysis and sequence alignment[END_REF] in the youngest cohorts exanthemata of viral or other origin 1 could be confused frequently with rubella infection[START_REF] Guy | Mumps 14 and rubella: a year of enhanced surveillance and laboratory testing[END_REF]].2The pattern of this epidemic was somewhat unusual. The outbreak epicentres were 3 two residential homes for the disabled, and this wave was stopped by vaccination of 4 susceptible subjects. Subsequently, rapid involvement of the general population 5 occurred. Considering the high rate of asymptomatic rubella infections, it is 6 conceivable that the circulation of the virus was extensive. Another epidemiological 7 feature was the marked age-shift of infection. Children were affected very rarely, 8 whereas the age group most affected was that of 20-29 years. Overall, the proportion 9 of females infected was low, probably owing to previous selective vaccination by 10 gender. A different exposure by gender could be excluded, at least in schools. The 11 pattern is consistent with that described in other European countries where rubella12 vaccination was offered to pre-pubertal girls before universal child immunisation and 13 where opportunistic rubella screening and vaccination are currently offered to women 14 of childbearing age [Jin and Thomas, 2007; Nardone et al., 2008; Schmid et al., 15 2009]. Despite the low proportion of infected women of childbearing age, three 16 pregnant women were infected, two voluntary abortions were performed and one 17 rubella virus was detected. A revision of CRS registry of this region did not show any 18 additional case of CRS or primary rubella infection in pregnancy until June, 2009. 19 Genotyping of the viruses isolated showed that the outbreak was sustained by a strain 20 classified as RUBV 2B genotype virus. Genomic characterisation was performed on 21 sixteen isolates by amplification of a screening window of 256 nt inserted in the E1 22 suggested by WHO Standardisation Committee [WHO, 2005b]. Although 1 the length of amplification fragments was sub-optimal in comparison to that 2 proposed, it has been demonstrated that this way of genotyping is still reliable [Jin 3 and Thomas, 2007]. Phylogenetic analysis showed that the rubella viruses isolated 4 during the present outbreak were related to the reference strain 5 RVi/Seattle.USA/16.00. Although the viruses circulating in both the care facilities 6 and the general population proved to be similar, being identical in all but one 7 aminoacidic sequences, the nucleotide sequences presented two synonymous 8 mutations apparently with a non-casual pattern. The CC pattern was significantly 9 more frequent in patients in institutions during the first half of the epidemic while the 10 TT pattern was present in community acquired rubella during the second wave. This 11 space-temporal pattern may be justified by an evolution of the virus during the 12 epidemic but intermediate CT or TC sequences were not found and a simultaneous 13 mutation in both sites seems unlikely. Thus, molecular tracing by sequencing 14 suggests an independent and subsequent introduction of two distinct variants of 15 RUBV genotype 2B. WHO Laboratory Network for measles and rubella viruses 16 surveillance reported the global distribution of RUBV genotypes, showing that in 17 Europe genotype 1E and 1G are predominant [WHO, 2006; Hubschen et al., 2007]. 18 More recently, the circulation of genotype 1j was documented in Spain, possibly 19 introduced by persons from Latin America [Martinez-Torres et al., 2009]. Genotype 20 2B is autochthonous in the Far East, namely Japan and Korea, and in South Africa, 21 although it is now considered an imported European genotype [Jin and Thomas, et al. 2009]. In Italy, genotyping of RUBVs isolated during three 1 consecutive outbreaks (1991-97) showed that clade 1 viruses were predominant, 2 while only two strains belonging to clade 2 were detected during the epidemic of 3 1994 [Zheng et al., 2003]. Interestingly, 2B genotype sequences detected in this study 4 clustered with one of these old isolates, suggesting an autochthonous origin of this 5 outbreak. Unfortunately, no temporal series of Italian rubella genotypes is available 6 to support this hypothesis. Otherwise, genotype 2B may have been imported, either 7 from countries where this virus was circulating or from the United Kingdom where 8 the genotype had been endemic since 2006. An interchange between Italy and the 9 United Kingdom has been demonstrated for rubella virus as well as for measles virus Ansaldi et al., 2009]. 11 The epidemic described could be attributed mainly to the vaccine cover in children, 12 still suboptimal, and due to the high rate of seronegative young adults, particularly 13 adult males. In this region, the current MMR vaccine cover of children at two years is 14 about 90%, thus missing the target rate of 95%. The offer of the second dose of the 15 MMR to children aged six-twelve years was launched in this region from 2003 and it 16 is estimated that the current cover based on the 2001 cohort is around 80%. The rate 17 of seronegative adults, notably women of childbearing age, is not well known. 18 Several efforts have been made in Europe to establish a standardized method for 19 attaining comparable data on seroepidemiology [Tischer et al., 2007]. A recent multi-20 country European survey showed that in several areas the proportion of rubella 21 immune women of childbearing age was far from the optimal figure of more than 22 the WHO. Twelve out of seventeen countries studied did not attain 1

9 with a marked

 9 difference between northern and southern areas of Italy. This figure 10 should be compared with that obtained by a cross-sectional survey carried out in 11 2006, just before the epidemic, and involving more than 1400 women of childbearing 12 age coming from a limited urban area of the same region. The data show that the 13 required proportion of rubella susceptible women (less than 5%) is now close to 14 being reached. Hence, apart from the need to improve universal vaccination by the 15 two-dose strategy, efforts should be made to identify seronegative women for

Fig. 2 .

 2 Fig. 2.-Distribution of confirmed rubella cases by month and origin.

Fig. 3 .

 3 Fig. 3. Phylogenetic tree of the 280 nt Rubella E1sequences obtained in the study (in bold), in comparison to reference viruses [WHO, 2007] and sequences obtained from GenBank. Reference strains and sequences from GenBank are indicated with the accession number followed by the strain name.

  Table III -Seroprevalence of Rubella IgG antibody (≥10 UI/ml) in women of childbearing age of Trieste. Fig. 1-Distribution of notified and confirmed cases of rubella by age.

	Page 21 of 22		Journal of Medical Virology Journal of Medical Virology				Page 20 of 22
	Tab. II -Epidemiologic and molecular features of 16 rubella strains representative of the outbreak
	Rubella IgG positive Source Genotype Institution 2B Institution 2B Institution 2B Institution 2B General pop. 2B Institution 2B General pop. 2B Institution 2B Institution 2B General pop. 2B General pop. 2B General pop. 2B N. % 26 92.9 129 93.5 280 95.2 454 94.0 365 95.3 84 93.3 1338 94.5 F o r P e e r R e v i e w Age group N. tested Patient age Sex Exanthema onset 25 F 28/01/08 30 M 28/01/08 38 M 30/01/08 29 M 03/02/08 34 M 04/02/08 57 F 16/02/08 15 M 19/02/08 30 M 21/02/08 54 F 01/03/08 31 M 22/03/08 28 M 03/04/08 14 M 03/04/08 15-19 28 20-24 138 25-29 294 30-34 483 35-39 383 40-44 90 Total 1416 r Rubella strain RVs/ FVG.ITA/05.08 RVi/ FVG.ITA/05.08/2 RVi/ FVG.ITA/06.08/1 RVi/ FVG.ITA/06.08/2 RVi/ FVG.ITA/06.08/3 RVi/ FVG.ITA/08.08 RVi/ FVG.ITA/09.08/2 RVi/ FVG.ITA/09.08/1 RVi/ FVG.ITA/10.08 RVi/ FVG.ITA/13.08 RVi/ FVG.ITA/15.08 RVi/ FVG.ITA/19.08 F o P e	Mutations* 2884 a 2904 b 2955 b T C C T C C T C C G C C T T T T Y Y T C C T C C T C C T T T T T T T T T
	RVi/ FVG.ITA/17.08 RVi/ FVG.ITA/21.08/1 RVi/ FVG.ITA/21.08/2 RVi/ FVG.ITA/21.08/3	1 15 21 e r 24 R F M F M	20/04/08 21/05/08 21/05/08 23/05/08	General pop. General pop. General pop. General pop.	2B 2B 2B 2B	T T T T	T T T T	T T T T
	* nucleotides are indicated according to the numeration of the rubella ORF2 sequence coding for structural protein; e a non synonymous mutation F962G, b synonymous mutations v i e
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Acknowledgements 19

We are very grateful to Mrs. Nadia Giannini and Giusi Carnelos who collected

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