Miho ; Yamaki
email: fac@tandf.co.uk

 1997). These measures check residues in milk and dairy products, and establish the frequencies and level of sampling, and the groups of substances to be controlled.

In Spain, ewes milk is monitored for antibiotic residues at dairy laboratories by several microbiological screening tests to control the presence of antibiotic residues. Some of these tests are the BRT  AiM, the Delvotest SP  or the Eclipse "100ov"  ( [START_REF] Barbosa | Suitability and Application of Available Test Kits for the Detection of Residues of Antimicrobials in Milk from Species Other than the Cow -a Review[END_REF]). The detection limits for the main antibiotics used in ovine therapy have been estimated in ewe milk for all these tests [START_REF] Althaus | Detection limits of antimicrobials in ewe milk by Delvotest[END_REF], Molina et al. 2003a[START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF].

The purpose of our study was to find out the incidence of antimicrobial residues in ewe milk destined to Manchego PDO cheese production by using the Eclipse "100ov"

Materials and methods

Milk sample collections

Ewe milk samples from bulk tanks were collected on a monthly basis from Manchega ewe flocks which supplied milk for PDO Manchego cheese production, from July 2002 to June 2003. A total of 2686 samples were collected in a 100 mL-disposable plastic container without any preservatives and kept at 4ºC until they were analysed (no later than 48 hours).

Another sample of milk was collected with 4 µl/mL solution of acidiol (150 mg chloranphenicol, 1 mL ethanol, 3.5 g sodium amide, 4.5 g sodium citrate 5H 2 O, in 100 mL of distilled water) for chemical, bacteria and SCC determinations. Milk bacteria and SCC levels were determined with a Fossomatic 90 (Foss Electric, Hillerød, Denmark) and a BactoScan 8000S (Foss Electric, Hillerød, Denmark), respectively. Chemical composition was determined with a MilkoScan FT 120 (Foss Electric, Hillerød, Denmark). Milk samples were analysed over a 48-hour post-collection period by the Eclipse "100ov"  test (ZEU-Inmunotec, Zaragoza, Spain). The method was carried out according to the manufacturer's instructions. The detection limits for the main antibiotics used in dairy ewes have been reported by [START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF]. Antibioticfree milk samples were used as "negative controls", and were obtained from controlled animals that did not receive any antibiotic treatment or feed for one month. As "positive controls", milk samples with 4 µg benzyl penicillin G (Sigma Chemical Co., MO, USA) kg -1 were used. In all tests visual interpretations were carried out by 3 professionals and evaluated as "negative", "doubtful" or "positive" qualifications. For the statistical calculations, the visual results that presented at least two similar interpretations were considered. The "positive" and "doubtful" responses were grouped as "positive and doubtful" [START_REF] Suhren | Detection of β-lactam antibiotics in milk by the Penzym-test[END_REF] in order to treat these qualitative variables at two levels:

Antibiotic Microbiological Screening Test

"negatives" and "positives and doubtfuls" (from now "positives"). All samples were simultaneously assayed by the Delvotest SP  method as described by Yamaki et al.

( 2004).

"Positive" samples were kept refrigerated at 4ºC and 24 hours later were heated at 82ºC for 10 min (Molina et al. 2003b) for confirmation.

Identification of the antibiotics

To identify the presence of β-lactam or sulphonamide compounds "positive" samples after heat confirmation they were tested with the penicillinase and the p-aminobenzoic acid (PABA) solutions. Four 100 µl-aliquots of each prepared sample were added to four wells of Eclipse "100ov"  test, followed by: 10 µl of distilled water, 10 µl of penicillinase solution (100,000 IU mL -1 ; Cod. 9120, AiM-Analytik in Milch Produktions-und Vertriebs-GmbH, München, Germany), and 10 µl of PABA solution (3 mg mL -1 ; Cod. A-9878, Sigma Chemical Co., MO, USA) respectively. Data interpretation was performed according to the combination of positive and negative results obtained for each series of four wells as mentioned by Yamaki et al. (2004).

Statistical analysis

To evaluate the influence of the numerical variables of the month of collection, fat, protein, SCC and bacteria on the responses to the Eclipse 100ov test a stepwise procedure was applied to the logistic regression option of Statgraphics Plus 5.1., taking the model of the variables that presented a value of Chi-squared ≥ 3.94. to be relevant.

The statistical design was carried out with the following logistic model:

L ijklmnop = β 0 + β 1 T i + β 2 T 2 j + β 3 T 3 k + β 4 T 4 l + β 5 SCC m + β 6 BAC n + β 7 F o + β 8 P p + ε ijklmnop
where L ijklmnop is the variable logit, i.e. ln P ijklmnop / 1-P ijklmnop ; P ijklmnop is the probability of "positive and doubtful" response; 1-P ijklmnop is the probability of "negative" response; β 0 to β 8 are coefficients estimated for the logistic regression models; T i is the effect of month (from 1 to 12); T 2 j is the effect of month squared; T 3 k is the effect month cubed; T 4 l is the effect of month quartered; SCC m is the effect of somatic cells; BAC n is the effect of bacteria; F o is the effect of fat; P p is the effect of protein; and ε ijklmnop is the residual error.

A Chi-squared Goodness of Fit Tests was conducted as a rank of correlation between the observed responses and the predicted probabilities.

In order to compare the results obtained by both tests, Delvotest SP and Eclipse 100ov, a cross tabulation option from the Statgraphics Plus 5.1 was done and the Chisquared test was carried out.

Results

During the year of the antimicrobial residue control, the incidence of "positives"

("positive and doubtful" responses) after the initial analysis by Eclipse "100ov"  was 2.6%. Study by means of a logistic regression model to evaluate the effects of factors such as the seasonality, the composition, the SCC or the bacteriology on test results was used.

The application of the logistic regression model only considered the effect of the month of collection to be significant (table 1). The prediction model for calculating the frequencies of "positive" samples, taking into account the month of collection was:

L= -2.8813 -0.1028 T 2 + 0.0159 T 3 -0.00064 T 4

The results of the Goodness-of-Fit Test for this logistic regression model showed a Chisquared of 1.7236; p= 0.6317, as the value p is superior to 0.1, there is no reason to reject the adaptation of the adjusted model to a level of confidence of 90% and over.

Figure 1 represents the relative frequencies of "positive" responses by the Eclipse "100ov"  test and the percentages of "positive" cases calculated by using the coefficients obtained with this logistic model. In this figure the highest frequencies can be seen in summer and early autumn. Samples which were "positive" after the initial analysis were confirmed by heating at 82ºC for 10 min. After this heat treatment the outcome of "positives" was 0.9%. As with the initial assay, the effect of the month collection variable was now the only significant variable (table 2). The mathematic The Goodness of Fit Test showed a Chi-squared of 4.3429 and p= 0.2267 (90% of confidence interval). By applying the logistic regression model (figure 1) the highest frequencies of "positive" results were presented in September and October.

The twenty-six samples that were confirmed after heating as "positive" were analysed by the tests of the penicillinase and PABA solutions (table 3). Most of them (19 samples) did not react positively with any of the two assessments, six samples reacted positively to penicillinase solution, and one sample could not be identified because of a dilution effect.

The second part of this work aims to compare the results obtained by the Eclipse "100ov" test with those obtained by the Delvotest SP  method, a method recognised by the Association of Official Analytical Chemists (AOAC 2004). Table 4 shows the results after the simultaneous analysis of raw milk samples by both methods. At the initial assay, a total of 101 samples were "positive" to Delvotest SP  whereas only 71

were "positive" to Eclipse "100ov"  , of which only 46 were "positive" in both tests.

The heat treatment reduced the number of "positive" samples to 47 and 26 for Delvotest SP  and Eclipse "100ov"  , respectively (table 5). But, only 17 samples were "positive" in both methods. The two analyses (initial and after heating) showed a higher detection of "positive" samples by the Delvotest SP  method than by the Eclipse "100ov"  . On the other hand, of the 17 samples that were "positive" by both methods, 10 samples corresponded to unidentified samples, 6 samples were "positive" to penicillinase solution and 1 sample was associated with a dilution effect when penicillinase and PABA solutions were assayed by both methods (data not showed).

In both methods the effect of the heat treatment showed a high number of "positive" samples at the initial assay (54 for the Delvotest SP  and 45 for the Eclipse "100ov"  ), which turned negative after heating (from now on "false positives"). When the "positives" and "false positives" that were detected simultaneously by the two methods were compared, only 5 coincided with a "false positive" result (table 6). Relative frequencies of "false positives" observed by both methods are shown in figure 2.

Discussion

After the heat treatment of the samples, the ratio of positives detected by Eclipse "100ov"  test in this study lowered to 1%. The control of the same samples by the Delvotest SP  showed a slightly higher level (1.7%; Yamaki et al. 2004). With regards to these levels of antimicrobial residues, the quality of ewe milk destined for the production of Manchego cheese with PDO seems to be more suitable than the ones reported by other studies in ewe milk (Esnal et al. 2002, Continanza et al. 2003, Herrera et al. 2004) Association (Gallego 2002). This fact could have had an important influence on a better sanitary status of the mammary gland by reducing the use of antibiotic therapy, and consequently, the number of residues in ewe milk. However, this incidence is still almost ten fold more than in cow milk (FDA/CFSAN 2001, Suhren and Reichmuth 2003), and sampling plans to control residues in ewe milk may continue in order to reduce the presence of antimicrobial residues even more.

A lower number of "positives" were detected when the samples were assayed by the Eclipse "100ov"  , compared to the Delvotest SP  . The sensitivity of these tests is an important factor to be considered in order to interpret the lower level of residues detected by Eclipse "100ov"  . [START_REF] Althaus | Detection limits of antimicrobials in ewe milk by Delvotest[END_REF] and [START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF] have studied the sensitivity of both methods for several antibiotics in ewe milk. Because the Eclipse "100ov"  test shows worse detection limits than the Delvotest SP  for several antibiotics [START_REF] Althaus | Detection limits of antimicrobials in ewe milk by Delvotest[END_REF][START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF], it is probable that the Delvotest SP  detected positives samples which contained lower concentrations of some antibiotics. According to these authors, sulphonamides or tetracyclines showed similar detection limits for both methods, while some antimicrobial groups such as β-lactams, aminoglycosides or macrolides could perhaps be underestimated when ewe milk is assayed by the Eclipse "100ov"  [START_REF] Althaus | Detection limits of antimicrobials in ewe milk by Delvotest[END_REF][START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF]. Six of the samples confirmed as positives by the Eclipse "100ov"  were identified by means of the penicillinase solution as β-lactams (table 3), and these samples were also identified Eclipse "100ov"  (6), probably due to a higher sensitivity for these compounds [START_REF] Althaus | Detection limits of antimicrobials in ewe milk by Delvotest[END_REF], even under the legal limits established by the European Union normative. On the other hand, there are a great number of "positive" samples by Eclipse "100ov"  (table 2) that were not identified as β-lactams or sulphonamides. Therefore, aminoglycosides, macrolides, tetracyclines, quinolones, or other antimicrobials different to β-lactams or sulphonamides could be presented in this group. Esnal et al. (2003) detected β-lactams, tetracyclines and macrolides by using a microbiological multiplate system to confirm residues in ewe milk. According to these authors these substances are commonly used for ovine mastitis therapy in Spain. For this reason it could be interesting to go more deeply into the detection of these substances using chromatographic methods (HPLC or HPLC-Mass Spectrometry), that are able to detect and correctly quantify these substances.

On the other hand both methods showed a high number of "false positives" even though from the point of view of percentages, the number of "false positives" detected by the Eclipse "100ov"  method was higher than those observed with the Delvotest SP  , as 63% of the positive samples of the Eclipse "100ov"  turned out to be "false positives" and 53% in the case of the Delvotest SP  method. Several authors have related "false positives" with the presence of natural inhibitors, SCC, micro-organisms, or free fatty acids (Carlsson et al. 1989, Van Eenennaam et al. 1993[START_REF] Andrew | Evaluation of selected antibiotic residue screening tests for milk from individual cows and examination of factors that affect the probability of false-positive outcomes[END_REF], Kang and Kondo 2001). Some naturally occurring compounds, such as the lactoperoxidase system, the lactoferrin or the lysozyme, could have an important negative effect on the Wolfson and Summer 1993). The enzymatic nature of these compounds favours their inactivation by heating, and consequently the diminution of erroneous responses. The effect of heating on the response of Delvotest SP  has been previously reported in cow and ewe milk (Kang and Kondo 2001, Molina et al. 2003b). The same took place with this method. The heat treatment of ewe milk at 82°C for 10 min decreased the number of positive samples detected by Eclipse "100ov"  test. This fact suggests the need to heat samples previous to analysis. However, these samples could also contain antimicrobial thermosensitive substances which justified this reduction [START_REF] Moats | The effect of processing on veterinary residues in foods[END_REF], Zorraquino et al. 2005).

In this trial, antimicrobial residue violations seemed to occur more in late summer and early autumn. The main results of the simultaneous assay with the Delvotest SP  test have been published by Yamaki et al. (2004), and showed that a significant higher incidence has been also observed in these months (Yamaki et al. 2004), mainly with regards to "false positives". This fact has been related to out-of-season breeding practices on Manchega ewes. The Manchega breed showed a lower seasonality in its reproductive performance, this allows more reproductive periods during the year.

Generally, the months of July and August coincided with the last part of the ewes' lactation period, at this time the content of natural inhibitors and SCC is higher (Gonzalo et al. 1994[START_REF] Bergonier | New advances in epizootiology and control of ewe mastitis[END_REF], which could provoke a higher level of "false positives".

The highest level of "positives" was observed from July to November. This period coincides with the final lactation of the animals who gave birth in January and February and when the milking period of out-of-season breeding animals starts. In these periods the milk production is lower with a concentration effect taking place in the milk (Gallego 2002;[START_REF] Pérez-Guzmán | Recuentos de células somáticas en leche de tanque de ganaderías de raza ovina Manchega durante los años 1992-1999[END_REF], this probably favours the concentration of antibiotics in the milk. In this study a lower level of milk production was observed (data not shown), which could explain the high number of positives detected.

The Eclipse "100ov"  is a screening test based on microbial inhibition principles which has been developed for assaying ewe milk. The number of positives detected with this test was lower than those observed with Delvotest SP  test, as much in the initial analysis (table 4) as in the confirmation after heating (table 5). [START_REF] Sischo | Field trial of four cowside antibiotic screening test[END_REF] defined the term "specificity" as "the relation between the number of negative results

and the total number of samples analysed by means of a determined method, using residue-free milk", and related it with "false positive" results. The specificities for Delvotest SP  and Eclipse "100ov"  methods were previously estimated at 97.7% and 99%, respectively (Molina et al. 2003b;[START_REF] Montero | Detection of antimicrobial agents by a specific microbiological meted (Eclipse 100  ) for ewe milk[END_REF].

Factors such as the chemical composition, physiological aspects, or hygienic-sanitary parameters (SCC and microbial level), could have an important influence on the detection of "false positive" results by microbiological tests [START_REF] Althaus | Accuracy of BRT and Delvotest microbial inhibition tests by composition of ewes milk[END_REF], Molina et al. 2003b). In this study, none of these factors seemed to interfere in the response of the Eclipse "100ov"  test, as the logistic regression results show (tables 1 and 2). By contrast, other tests suffer more interference due to some of these factors, which have been observed in other studies carried out in ewe milk [START_REF] Althaus | Accuracy of BRT and Delvotest microbial inhibition tests by composition of ewes milk[END_REF], Molina et al. 2003b, Yamaki et al. 2004). This test has been specifically developed to be used in ewe milk, and some interference due to the intrinsic or extrinsic characteristics of ewe milk could have been solved by the manufacturer. Therefore, the detection of "false positive" cases should be lower in ewe milk. However, a slightly higher number of "false positives" has been detected with the Eclipse "100ov"  method which could be attributed to a greater sensitivity when faced with natural inhibitors.

This factor has not been analysed in this study.

Conclusions

In general, the Eclipse "100ov"  method detected a slightly lower number of positive samples than the Delvotest SP  and a slightly higher number of "false positives". The results obtained by the Eclipse "100ov"  test indicated that approximately 99% of the ewe milk assayed in Castilla-La Mancha region (Spain) destined to Manchego PDO cheese production is appropriate for human consumption. Regardless of the seasonality, none of the variables studied (composition, SCC or bacteriology) seemed to have an influence on the response of this method. Among the positive samples, 25% corresponded to β-lactam substances, but a preponderance of unidentified substances was appreciated. Therefore, it is necessary to make an effort to increase the knowledge of the presence of residues in ewe milk at a screening level to guarantee food safety.

F

  the frequency of ''positive'' cases for the Eclipse "100ov"  method was: L= -9.4844 + 4.131 T -1.0083 T 2 + 0.0928 T 3 -0.00288 T 4

  which showed higher percentages of positive cases. Manchego cheese is the most important Spanish PDO cheese(MAPYA 2002b), and the strict control of milk destined to its elaboration has to be the first requirement to guarantee, not only certification, but also a safe product. The hygienic-sanitary quality of Manchega ewe milk has undergone improvements over the last few years[START_REF] Pérez-Guzmán | Recuentos de células somáticas en leche de tanque de ganaderías de raza ovina Manchega durante los años 1992-1999[END_REF], mainly assisted by the regular monitoring of some hygienic-sanitary parameters (SCC ) carried out by the sanitary authorities and the Manchega Breed

  as β-lactams by the Delvotest SP  test. The number of samples identified as β-lactams by the Delvotest SP  (14;Yamaki et al. 2004) was higher than those observed by the

  growth of the bacteria indicators used in microbiological assays by showing "false positive" results, mainly due to their antimicrobial activity(Carlsson et al. 1989,

Table 1 .

 1 Summary of the logistic regression model coefficients for the initial analysis byEclipse "100ov"  test.

	Parameter	"χ 2 " value	"p" value
	Month 1

Table 2 .

 2 Summary of the logistic regression model coefficients for the confirmation afterheating at 82ºC 10 min by Eclipse "100ov"  test.

	Parameter	"χ 2 " value	"p" value
	Month 1

Table 3 .

 3 Identification of "positive and doubtful" samples (absolute and relative frequencies) after the PABA and penicillinase tests.

	Compound	Absolute frequency	Relative frequency (%)
	Non Identified Substance	19	73
	β-lactam	6	23
	Sulphonamide	-	-
	β-lactam and sulphonamide	-	-
	Diluted Samples	1	4
	Total	26	100

Table 4 .

 4 Data comparison of Eclipse "100ov"  results with Delvotest SP  * results after initial analysis.

	Results	Eclipse"100ov"  Positives Negatives	Total	"χ 2 "	"p" value
		Positives	46 (1.71%)	55 (2.05%)	101 (3.76%)
	Delvotest SP 	Negatives	(0.93%) 25	(95.31%) 2560	(96,24%) 2585	750.56	0.0000
		Total	71 (2.64%)	2615 (97.36%)	2686 (100%)

* Data for comparing from

Yamaki et al. (2004)

.

Table 5 .

 5 Data comparison of Eclipse "100ov"  results with Delvotest SP  * results after confirmation analysis after heating at 82ºC 10 min.

	Results	Eclipse"100ov"  Positives Negatives	Total	"χ 2 "	"p" value
		Positives	17 (0.63%)	30 (1.12%)	47 (1.75%)
	Delvotest SP 	Negatives	(0.34%) 9	(97.92%) 2630	(98,25%) 2639	644	0.0000
		Total	26 (0.97%)	2660 (99.03%)	2686 (100%)

* Data for comparing from

Yamaki et al. (2004)

.

Table 6 .

 6 Data comparison of Eclipse "100ov"  with Delvotest SP  * positive and false positive results.Figure 1. "Positive and doubtful" rate (%) in ewe milk samples at the initial analysis and at the confirmation analysis after 24 hours (heated at 82°C for 10 min) by Eclipse "100ov"  from July 2002 to June 2003. (•) Real values. ( -) Predicted values.Figure 2. Monthly relative frequencies (%) of "false positive" samples by the Delvotest SP ® ( ; n= 54) and Eclipse "100ov" ® ( ; n=45) methods.

			Eclipse"100ov" 
	Results	Positives	False Positives	Total	"χ 2 "	"p" value
		Positives	17 (36.96%)	19 (41.30%)	36 (78,26%)
	Delvotest SP 	False Positives	5 (10.87%)	5 (10.87%)	10 (21.74%)	17.93	0.0000
		Total	22 (47.83%)	24 (52.17%)	46 (100%)

* Data for comparing from

Yamaki et al. (2004)

.

Screening of antibiotic residues in ewe milk destined to cheese by a commercial microbiological inhibition assay

	Introduction	Abstract
	Bulk ewes milk from Spanish dairy farms situated in the Castilla-La Mancha region, Currently, Spain is the third most important ewe milk producer of the European
	and destined for production of Protected Denomination of Origin (PDO) Manchego Union-25 with a turnover of about 16.6% (FAOSTAT 2004). This production is mainly
	cheese, were analysed each month for antimicrobial residues during the course of one focused in two regions: Castilla-León and Castilla-La Mancha. Of the two regions,
	year. For this study a microbiological assay specific for ewes milk (Eclipse "100ov"  ) Castilla-La Mancha takes second place (31.9%) and uses almost all this milk for cheese
	was used. The number of positive samples by the Eclipse "100ov"  test was 2.6%. A production (MAPYA 2002a). The Protected Denomination of Origin (PDO) Manchego
	F second examination of positives following heat treatment at 82ºC for 10 min showed a cheese is very important in this region, since it is the most produced Spanish PDO o r reduction of up to 0.9% (in other words 63% of the samples detected corresponded to cheese (43% of the total PDO cheeses; MAPYA 2002b).
	P e In order to ensure the hygienic-sanitary quality and safety of raw milk the EU
	established food regulations in the dairy sector (Council Directives 92/46/CEE of 16 e r June 1992, Council Directives 96/23/EEC of 29 April 1996; Commission Decision
	97/747/EC of 27 October	R
		e
	v i obtained by the Delvotest SP  method and showed the detection of a smaller number of e w positive samples with the Eclipse "100ov"  method.
		O n l
		y
	Key words: ewe milk quality, antibiotic residue, screening test, Eclipse "100ov" 

M. YAMAKI † ‡, M.I. BERRUGA † ‡*, R.L. ALTHAUS §, M.P. MOLINA# and A. MOLINA † ‡ †Departamento de Ciencia y Tecnología Agroforestal, ETSIA, Universidad de Castilla-La Mancha, 02071 Albacete, Spain ‡Seccion de Calidad Alimentaria, Instituto de Desarrollo Regional, Universidad de Castilla-La Mancha, 02071 Albacete, Spain §Cátedra de Biofísica, Facultad de Ciencias Veterinarias, Universidad Nacional del Litoral, 3080 Esperanza, Argentina #Departamento de Ciencia Animal, Universidad Politécnica, 46071 Valencia, Spain

*To whom correspondence should be addressed. e-mail: mariaisabel.berruga@uclm.es "false positives"). Twenty-five percent of the confirmed positives were identified as βlactams by a penicillinase solution, the remainder corresponded to antimicrobial compounds that could not be identified. The month of collection was related to the probability of positive results, but no other factors such as somatic cell counts (SCC), bacteriology or composition (fat and protein). The highest rates of positives were observed in September and October. Finally the results were compared with those

  CARLSSON, A., BJORCK, L., and PERSSON, K., 1989, Lactoferrin and lysozyme in milk during acute mastitis and their inhibitory effect in Delvotest P. Journal of .libroblancoagricultura.com/libroblanco/jautonomica/c_mancha/comunicacion es/gallego.pdf (accessed 12 January 2005). GONZALO, C., CARRIEDO, J.A., BARO, A., and PRIMITIVO, F.S., 1994, Factors influencing variation of test day milk yield, somatic cell count, fat, and protein in dairy sheep. Journal of Dairy Science, 77, 1537-1542. HERRERA, E., ABECIA, J.A., and MARTÍN, S., 2004, Antibióticos en leche de tanque. Albéitar, 75, 54-55. KANG, J-H., and KONDO, F., 2001, Occurrence of false-positive results of inhibitor on milk samples using the Delvotest SP assay. Journal of Food Protection, 64, 1211-SUHREN, G., and REICHMUTH, J., 2003, Messbarkeit und Entwicklung der hygienischen Wertigkeit des Rohstofs Milch. Kieler Milchwirtschaftliche Forschungsberichte, 55, 5-36. Van EENENNAAM, A.L., CULLOR, J.S., PERANI, L., GARDNER, I.A., SMITH, W.L., DELLINGER, J., and GUTERBOCK, W.M., 1993, Evaluation of milk antibiotic residue screening tests in cattle with naturally occurring clinical mastitis. Journal of Dairy Science, 76, 3041-3053. WOLFSON, L.M., and SUMMER, S.S., 1993, Antibacterial activity of the lactoperoxidase system: A review. Journal of Food Protection, 56, 887-892. YAMAKI, M., BERRUGA, M.I., ALTHAUS, R.L., MOLINA, M.P., AND MOLINA, A., 2004, Occurrence of antibiotic residues in milk from Manchega Ewe Dairy Farms. Journal of Dairy Science, 87, 3132-3137. ZORRAQUINO, M.A., 2005, Inactivación térmica de sustancias antimicrobianas en la leche. Tesis Doctoral. Universidad Publica de Navarra. España. 140 pp.

	Food Additives and Contaminants
	samples. The authors are also grateful to ZEU-Inmunotec (Zaragoza, Spain) for their FAOSTAT, 2004, Datos Agrícolas de FAOSTAT. Available online at:
	support. www.faostat.fao.org/faostat/collections?version=extandhasbulk=0andlanguage=ES
	Dairy Science, 72, 3166-3175. (accessed 10 December 2004),
	CONTINANZA, R.M., TIBURTINI, S., and PAGGI, U., 2003, Determinazione di FOOD AND DRUG ADMINISTRATION/CENTER FOR FOOD SAFETY AND
	inibenti nel latte mediante kit microbiologico. Il latte, 28 (4), 80-84. APPLIED NUTRITION, 2001, National Milk Drug Residue Data Base Fiscal Year
	EC, 1992, Council Directive (EC) 92/46 of 16 June 1992 laying down the health rules 2000 Annual Report, Available online at:
	o r F o r for the production and placing on the market of raw milk, heat-treated milk and milk-based products. Official Journal of the European Communities, L 268, pp. 18-F o r www.vm.cfsan.fda.gov/~ear/milkrp00.html (accessed 4 May 2004). GALLEGO, R., 2002, Análisis de estructuras y sistemas de producción en el sector del F o r F o r
	P e P e P ovino Manchego. P P EC, 1996, Council Directive (EC) 96/23 of 29 April 1996 on measures to monitor 24. ganado Available online at: e e e
	e r e r certain substances and residues thereof in live animals and animal products and repealing Directives 85/358/EEC and 86/469/EEC and Decisions 89/187/EEC and e r e r e r
	R R 91/664/EEC. Official Journal of the European Communities, L 125, pp. 10-32. R R R
	e e EC, 1997, Commission Decision (EC) No 97/747 of 27 October 1997 fixing the levels e e e
	v i e w v i e w and frequencies of sampling provided for by Council Directive 96/23/EC for the v i e v i e v i e monitoring of certain substances and residues thereof in certain animal products. Official Journal of the European Communities, L 303, pp. 12-15. w w w
	O n l y O n l y ESNAL, A., GAMALLO, J.A., MARCO, J.C., RODRÍGUEZ, L., SOPENA, J., MARTINEZ, F., DEL REAL, M.A., GARCÍA, I., ORDÓÑEZ, J., MARTÍN, S., and O n l 1215. O n l O n l EXTRAMIANA, A.B., 2002, Valoración práctica de un estudio de inhibidores y de recuento de patógenos contagiosos en leche de tanque de cuatro agrupaciones de y MINISTERIO DE AGRICULTURA, PESCA Y ALIMENTACIÓN, 2002a, Anuario y y
	productores de leche de oveja. Proceedings of the VI Jornadas Internacionales de la de estadística agroalimentaria. Available online at:
	Sociedad Española de Ovinotecnia y Caprinotecnia, 19-21 September, Valencia, www.mapya.es/estadistica/Anu_02/pdf/PDF21_09.pdf (accessed 11 March 2004).
	Spain, edited by SEOC (Valencia, Spain), pp 576-582.

wwwMINISTERIO DE AGRICULTURA, PESCA Y ALIMENTACIÓN, 2002b, Datos de las Denominaciones de Origen Protegidas (D.O.P.) e Indicaciones Geográficas Page 25 of 27 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.uk

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

Acknowledgements

This work was funded by project 191/IA-40, from the Consejeria de Agricultura y Medio Ambiente (Junta de Comunidades de Castilla-La Mancha, Spain). The authors would like to thank the workers from the Forlactaria Operadores Lecheros S.A. dairy company (Villarrobledo, Albacete, Spain) for their valuable help in the collection of


Karen Seeling

Sven Dänicke

Hana Valenta

Hans Van Egmond

Ronald Schothorst

Peter Lebzien

Margit Schollenberger

Ebrahim Razzazi-Fazeli

A A Jekel

On the effects of Fusarium toxin contaminated wheat and feed intake level on biotransformation

Keywords: Mycotoxins -fusarium, Mycotoxins -deoxynivalenol Food Types: Milk, Animal feed Deoxynivalenol, Fusarium, ruminants, level of feed intake, milk, carry over 53 54

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

On the effects of Fusarium toxin contaminated wheat and feed intake level on 1 the biotransformation and carry over of deoxynivalenol in dairy cows 2 3 4 K. SEELING 1* , S. DÄNICKE 1 , H. VALENTA 1 , H. P. VAN An experiment was carried out to examine the effects of feeding Fusarium toxin contaminated 21 wheat (8.21 mg deoxynivalenol, DON, and 0.09 mg zearalenone, (ZON), per kg dry matter, 22 DM) at different feed intake levels on biotransformation and carry over of DON in dairy 23 cows. For this purpose, fourteen ruminal and duodenal fistulated dairy cows were fed a diet 24 containing 60 % concentrate with a wheat portion of 55 % (Fusarium toxin contaminated 25 wheat [mycotoxin period] or control wheat [control period]) and ration was completed with 26 maize-and grass silage (50:50) on a DM basis. Daily DON intakes ranged from 16.6 up to 27 75.6 mg in the mycotoxin period at DM intakes of 5.6 up to 20.5 kg. 28 DON was almost completely biotransformed to independent of the 29 DON/feed intake, and the flow of DON and de-epoxy DON at the duodenum related to DON 30 intake ranged between 12 and 77 % when the Fusarium toxin contaminated wheat was fed. In 31 the serum samples, de-epoxy DON was detected in the range of 4 up to 28 ng/ml in the 32 mycotoxin period, while concentrations of DON were all below the detection limit. The daily 33 excretion of DON and de-epoxy DON in the milk of cows fed the contaminated wheat varied 34 between 1 and 10 µg and 14 and 104 µg, respectively. The total carry over rates as the ratio 35 between daily excretion of DON and de-epoxy DON into milk and DON intake were in the 36 Introduction 55 Deoxynivalenol (DON) is one of the most frequently recognised mycotoxins in Europe 56 produced by several Fusarium species on a variety of cereal grains (Bottalico and Perrone 57 2002). In animal livestock, ingestion of DON can cause numerous adverse toxicological 58 responses associated with reduced performance and immune function (Rotter and Prelusky 59 1996). In contrast to pigs, which react most sensitively to DON, ruminants appear to be 60 relatively tolerant, which is attributed to the metabolisation of the toxin by microbes in the 61 rumen. Several in vitro studies with bovine rumen fluid have demonstrated that DON is 62 biotransformed extensively to the less toxic compound de-epoxy DON [START_REF] King | Transformation of 517 deoxynivalenol (vomitoxin) by rumen microorganisms[END_REF], 63 Swanson et al. 1987[START_REF] He | Microbial transformation of deoxynivalenol 511 (vomitoxin)[END_REF]. Consequently, the diversion of cereal batches suspected 64 to be contaminated with DON to ruminants is a useful means of utilising grain that is 65 unsuitable for humans or monogastric farm animals. But, in combination with higher DON 66 concentrations in cereals and roughages during so called Fusarium years (Oldenburg et al. 67 2000), the influence of DON on the health and performance of ruminants should not be 68 underestimated. However, little research has been published on the effects of DON on 69 ruminants. Most of the reports were given as field or case studies and experiments under 70 3 controlled conditions were only performed with small numbers of animals using either 71 purified DON or naturally contaminated feedstuffs (for reviews see [START_REF] Höltershinken | Mykotoxikosen beim Rind -513 Probleme auch in Norddeutschland?[END_REF], 72 Whitlow and Hagler 1999[START_REF] Bauer | Mykotoxine in Futtermitteln: Einfluss auf Gesundheit und Leistung[END_REF][START_REF] Dänicke | 503 Risikofaktoren für die Fusariumtoxinbildung in Futtermitteln und 504 Vermeidungsstrategien bei der Futtermittelerzeugung und Fütterung. Landbauforschung 505 Völkenrode[END_REF], Seeling and Dänicke 2005). 73 Thus, the described effects are quite variable between the different studies and it is difficult to 74 draw conclusions for the practical feeding. Furthermore, high-producing dairy cattle in early 75 lactation also appear more sensitive to DON than low-producing dairy cattle (Whitlow and 76 Hagler 1999). But no studies are available considering factors other than dosage and duration 77 of toxin exposure, which could cause these differences in susceptibility. Additionally, the 78 information about the carry over of DON and de-epoxy DON into the milk of dairy cows 79 feeding practically relevant concentrations are still rare. While no transmission of DON or de-80 epoxy DON to milk was observed in a study of [START_REF] Charmley | Influence of level of deoxynivalenol in 496 the diet of dairy cows on feed intake, milk production, and its composition[END_REF], trace amounts of 81 DON and de-epoxy DON could be detected by [START_REF] Prelusky | Transmission 528 of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF] and [START_REF] Cote | 499 Excretion of deoxynivalenol and its metabolites in milk, urine, and faeces of lactating 500 dairy cows[END_REF]. 82

Possibly, the feed intake (passage rate) as indicator for the feed retention in the rumen could 83 alter the ruminal metabolisation of DON. A rapid passage rate as an effect of increased feed 84 intake may limit the available time for a metabolisation of DON to de-epoxy DON in the 85 rumen. Due to a modified ratio of DON to de-epoxy DON at the duodenum, the toxicity as 86 well as the carry over of these substances in the milk could be altered. Therefore, the aim of 87 the present study was to examine the effect of varying levels of feed intake using Fusarium 88 toxin contaminated or non contaminated wheat on biotransformation and carry over of DON 89 by dairy cows. The paper is part of a comprehensive study where non contaminated control 90 wheat and Fusarium toxin contaminated wheat were fed to ruminants at different feed intake 91 levels. In further papers the influence on ruminal fermentation and milk composition as well 92 as the biotransformation of the mycotoxin ZON were described (Seeling et al. 2005a, Seeling 93 et al. 2005b). 94 95

Material and methods 96

Treatments, experimental design and animals 97

The experimental design and procedures were described previously (Seeling et al. 2005a, 98 Seeling et al. 2005b). In brief, fourteen dairy cows of the "German Friesian breed" equipped 99 with permanent ruminal and duodenal cannulas were selected for this experiment. The cows 100 had a mean live weight of 626 ± 71 kg. The animals were in different stages of lactation: 101 Cows 4-14 and Cows 5-14 lactated in the mycotoxin period and in the control period, 102 respectively, whereas the rest did not lactate. They were tethered using neck straps, housed in 103 All cows were fed a diet containing 60 % concentrate and 40 % maize-and grass silage 105 (50:50) on a DM basis (Table 1). The only cereal component of the concentrate was either 55 106 % uncontaminated control wheat (control period) or 55 % Fusarium toxin contaminated 107 wheat (mycotoxin period). Further components of the concentrate were 25 % soy bean meal, 108 16 % dried sugar beet pulp, 2 % soy oil and 2 % mineral feed. The composition of the dietary 109 ration was constant for all animals, only the daily amounts were adjusted individually 110 according to the current performance so that total dry matter intakes of the cows ranged from 111 5.6 up to 20.5 kg. Each cow was fed both the uncontaminated control wheat and the Fusarium 112 contaminated wheat in two subsequent periods. The concentrate was offered at 0500 h, 0800 113 h, 1500 h and 1800 h in equal-sized meals. Silages were given twice daily in equal portions at 114 0800 h and 1500 h. Cows were milked at 0500 h and 1500 h and milk yields were recorded at 115 each milking. Weights of the cows were recorded before and after the experimental periods. 116 117

Measurement and sample collection 118

Each experimental period lasted for four weeks. Following an adaptation period of three 119 weeks to the diets, duodenal samples were collected during the last week in order to estimate 120 the duodenal flow using chromium oxide (Cr 2 O 3 ) as marker [START_REF] Rohr | Measurement of duodenal flow 550 in dairy cows by either total collection or spot sampling, using a special cannula[END_REF]. The marker 121 was given in four boluses into the rumen. Marker administration started ten days prior to the 122 duodenal sampling period. Spot samples of duodenal chyme were taken at 2 h intervals from 123 Monday to Friday yielding a total of 60 samples for each cow. The whole procedure followed 124 the principles as described by [START_REF] Rohr | Der Einfluss eines 547 teilweise Ersatzes von Futterprotein durch Harnstoff auf den Stickstoff-und 548 Aminosäurenfluss am Duodenum[END_REF]. Blood samples (25 ml) were drawn from 125 each cow in each treatment one day in the 3rd week at 1000 h. Serum was separated by 126 centrifugation at 2000 x g for 20 min at 15°C 2 h after sampling and was kept at -20°C until 127 analysis. Milk sampling from each lactating cow took place through six consecutive milkings 128 in the third week of each period. The samples were pooled based on the weight of milk 129 produced at the morning and evening milking and frozen at -20°C, then freeze-dried and 130 stored at -20°C until further analysis. Faecal samples from the rectum as well as spontaneous 131 urine samples were collected in the last week of each experimental period. Faeces and urine 132

were immediately frozen at -20°C after sampling. Representative samples of the concentrates, 133 silages and feed refusals, if they occurred, were collected daily and pooled over the five-day 134 duodenal sampling trial. For proximate analyses, these samples were dried at 60°C and 135 ground to pass though a 1-mm screen. 136 Analyses of crude nutrients of feedstuffs were done on dry samples according to the methods 139 of the 'Verband Deutscher Landwirtschaftlicher Untersuchungs-und Forschungsanstalten' 140 (VDLUFA;[START_REF] Bassler | Die chemische Untersuchung von Futtermitteln, Methodenbuch. Bd. 484 3 mit Ergänzungslieferungen[END_REF]. After DM determination duodenal digesta and faeces were freeze-141 dried and ground to pass though a 1 mm sieve. Aliquots of the duodenal digesta from each 142 day were pooled according to daily DM flow calculated from Cr 2 O 3 concentration. Chromium 143 concentration in duodenal digesta and faeces was determined by atomic spectrophotometry 144 according to [START_REF] Williams | The determination of chromic oxide in 598 faeces samples by atomic absorption spectrophotometry[END_REF]. 145 DON in feedstuffs was determined by high performance liquid chromatography (HPLC) with 146 diode array detection (DAD) after extraction by acetonitrile-water (83:16, v/v) and clean up 147 with immuno-affinity columns (IAC; DONtest HPLC columns, VICAM, Watertown, USA) 148 according to a modified VDLUFA method as described by [START_REF] Valenta | Vergleich einer HPLC-und einer ELISA-586 Methode zur Bestimmung von Deoxynivalenol in Mühlenstäuben, Kleien und Getreide. 587 VDLUFA-Kongressband[END_REF]. The 149 detection limit was 0.03 mg/kg. The mean recovery for feedstuffs was 74 %. ZON in 150 feedstuffs was analysed by HPLC after incubation with 2 U β-glucosidase (E. C. 3.2.1.21;No. 151 G-0395, Sigma, Taufkirchen, Germany) according to [START_REF] Ueberschär | Einfluß von Zearalenon auf Wachstum und Rückstände in den 583 Geweben von Mastkaninchen[END_REF]. The mean recovery 152 was 86 %. The detection limit was 1 ng/g. Determination of the trichothecenes (nivalenol 153 (NIV), scirpentriol, T2-tetraol, fusarenon-X (FUS-X), monoacetoxyscirpenol (MAS), 15-154 acetyldeoxynivalenol (15-AcDON), 3-acetyldeoxynivalenol (3-AcDON), T2-triol, 155 neosolaniol (NEO), diacetoxyscirpenol (DAS), HT-2 toxin (HT2) and T-2 toxin (T2)) was 156 carried out according to a gas chromatography-mass spectrometry (GC-MS) method as 157 described by [START_REF] Schollenberger | Survey of Fusarium toxins in foodstuffs of plant origin marketed in Germany[END_REF]. 158

Freeze-dried duodenal digesta and faeces were analysed for DON and de-epoxy DON by 159 HPLC-DAD after incubation with β-glucuronidase, extraction with acetonitrile-water and 160 clean up by immuno-affinity columns (IAC; DONtest HPLC columns, VICAM, Watertown, 161 USA) according to [START_REF] Valenta | Determination of deoxynivalenol and de-589 epoxy deoxynivalenol in animal tissues by liquid chromatography after cleanup with an 590 immunoaffinity column[END_REF]. Serum and urine samples were analysed for DON 162 and de-epoxy DON by HPLC-DAD after incubation with β-glucuronidase, extraction with a 163 ChemElut  cartridge (Varian, Darmstadt, Germany) by ethyl acetate and clean up with IAC 164 following a HPLC method by [START_REF] Valenta | Determination of deoxynivalenol and de-589 epoxy deoxynivalenol in animal tissues by liquid chromatography after cleanup with an 590 immunoaffinity column[END_REF]. In contrast to the described method by 165 [START_REF] Valenta | Determination of deoxynivalenol and de-589 epoxy deoxynivalenol in animal tissues by liquid chromatography after cleanup with an 590 immunoaffinity column[END_REF] incubation with β-glucuronidase was modified. Acetate buffer, pH 5.5, 166 was added to the faeces and urine samples and phosphate buffer, pH 6.8, was added to the 167 digesta and serum samples. After addition of β-glucuronidase (type H-2, min. 100000 U/ml, 168 sulfatase up to 7500 U/ml, Sigma-Aldrich, Taufkirchen, Germany), the mixture was incubated 169 overnight at 37 °C. The detection limits for both toxins were approximately 0.02 mg/kg DON and de-epoxy DON were determined to be 83 % and 92 % (duodenal chyme), 92 % and 172 86 % (faeces), 85 and 97 % (serum) and 88 and 88 % (urine), respectively. To confirm the 173 results in serum, four serum samples were incubated, extracted and purified as described 174 above and analysed by HPLC-mass spectrometry (HPLC-MS) according to Razzazi-Fazeli et 175 al. (2003). 176 Six contaminated serum samples (Cows 4, 6, 8, 10, 12 and14) andsix contaminated urine 177 samples (Cows 3, 4, 5, 9, 10, 14) of cows fed the contaminated wheat were parallelly 178 investigated with and without enzymatic (β-glucuronidase) digestion in order to estimate the 179 degree of glucuronidation of DON and de-epoxy DON in serum and urine. 180

Freezed dried milk samples were analysed for DON and de-epoxy DON at first without 181 incubation with β-glucuronidase by a GC-MS method after extraction by water/ethanol 182 (90/10), cleaning up by means of ChemElut  (Varian, Darmstadt, Germany) and Mycosep  183 columns according to [START_REF] Schothorst | Determination of trichothecenes in duplicate diets for young children by 562 capillary gas chromatography with Mass Spectrometric Detection[END_REF]. Trimetylsilyl (TMS) ether was used for 184 derivatisation and residues were dissolved in iso-octan. Beside DON and de-epoxy DON, the 185 trichothecenes FUS-X, NIV, 3-AcDON, 15-AcDON, DAS, NEO, HT2 and T2 in the milk 186 samples were determined with the same GC-MS method. The detection limits for DON, FUS-187 X, NIV, 3-AcDON, 15-AcDON, DAS, NEO, HT2 and T2 were 0.1, 0.16, 0.25, 0.22, 0.18, 188 0.30, 0.33, 0.22 and 0.16 µg/kg. The recoveries were determined to be 91,96,44,104,97,189 104, 109, 106 and 111 %. The samples were further analysed by HPLC-UV after incubation 190 with β-glucuronidase, extraction with acetonitrile-water and clean up by immuno-affinity 191 columns (DONprep, r-biopharm) according to the procedure described above for freeze-dried 192 faeces with slight modifications after detecting de-epoxy DON predominately as glucuronide 193 conjugate in the serum. The recoveries for DON and de-epoxy DON were determined to be 194 72 % and 89 %. The detection limits were 0.5 µg/kg. All milk samples of cows fed the 195 contaminated wheat were parallelly investigated without enzymatic (β-glucuronidase) 196 digestion in order to establish the degree of glucuronidation of DON and de-epoxy DON 197 using the HPLC-UV method. To confirm the results in the milk, the milk samples of all cows 198 fed the contaminated wheat were again analysed by the GC-MS method described above for 199 milk samples with enzymatic (β-glucuronidase) digestion. Acetate buffer, pH 5.2, was added 200 to the milk samples. After addition of β-glucuronidase (Helix Pomatia Juice from BioSepra; 201 activites > 100,000 FU/ml), the mixture was incubated overnight at 37 °C. 202

Results of mycotoxin analysis were not corrected for recovery. 203 [START_REF] Statsoft | Statistica for the Windows TM Operating System[END_REF]. 224

225 Results

226

Wheat and diets 227

The contents of crude nutrients and amino acids as well as the contents of analysed Fusarium 228 toxins for control and Fusarium contaminated wheat are summarised in Table 2. Only slight 229 differences in crude nutrients could be detected between control and contaminated wheat. The 230 Fusarium toxin contaminated wheat contained on average 8 mg DON and 0.09 mg ZON per 231 kg DM, while 0.25 mg DON and 0.05 mg ZON per kg DM were detected in the control 232 wheat. Maize silage which was used as roughage both in the control and in the toxin rations 233 also contained low levels of both toxins. In comparison with the control wheat, slightly higher 234 concentrations of 15-acDON, 3-acDON, scirpentriol and HT2 were detected in the Fusarium 235 contaminated wheat. 236 The daily flow of the sum of DON and de-epoxy DON at the duodenum ranged between 2.1 238 and 35.6 mg after feeding the Fusarium toxin contaminated wheat and between 0.4 and 4.4 239 mg after feeding the control wheat (Table 3). The predominant substance was de-epoxy DON 240 with a daily flow at the duodenum of 2.1 up to 35.1 mg and of 0.4 up to 4.0 mg in the 241 mycotoxin and control period, respectively, whereas the daily flow of DON varied between 242 0.04 to 2.2 mg as well as 0.01 to 1.3 mg. The flow of DON and de-epoxy DON at the 243 duodenum relative to the DON intake varied between 12 and 77 % when the Fusarium 244 contaminated wheat was fed and between 32 and 153 % in the control period. Thereby, the 245 portion of DON and de-epoxy DON of the total DON and de-epoxy DON flow were in the 246 range of 1 and 6 % as well as 94 and 99 % in the mycotoxin period (Figure 1) and between 3 247 and 35 % as well as 65 and 97 % in the control period, respectively. 248 249

Mycotoxin residues in serum 250

The metabolite de-epoxy DON was detected in all serum samples of the cows in the 251 mycotoxin period. The presence of de-epoxy DON was confirmed by HPLC-MS (Razzazi-252 Fazeli et al. (2003). Values ranged between 4 and 28 ng/ml (Figure 2). DON concentrations 253 were all below the detection limit. DON could not be detected in the serum samples of the 254 control period, whereas the concentrations of de-epoxy DON ranged between below the 255 detection limit and 5 ng/ml. Furthermore, based on the analysis of six samples with and 256 without incubation with β-glucuronidase, the degree of glucuronidation of de-epoxy DON 257 was found to be 100 %. It has to be stressed, however, that it is possible that due to the low 258 concentrations of conjugated de-epoxy DON in the serum samples a slight amount of non-259 conjugated de-epoxy DON could not be detected. 260 261

Mycotoxin residues in milk 262

Using the HPLC-UV method without β-glucuronidase incubation, de-epoxy DON was 263 determined in only one sample (Table 4), whereas the metabolite was detected in the milk of 264 all cows fed the contaminated wheat using the same method with β-glucuronidase incubation. 265

Based on the analysis of the one sample, the degree of glucuronidation of de-epoxy DON was 266 found to be approximately 77 %. DON was both with and without enzyme incubation in all 267 milk samples below the indicated detection limit using the HPLC-UV method. Concentrations 268 Both DON and de-epoxy DON were detected using the more sensitive GC-MS method 271 without and with enzyme incubation (Table 4). However, it has to be stressed that DON levels 272 were barely above the detectable limit. Values of the further analysed trichothecenes in the 273 milk were all below the detection limit. Concentrations of DON and de-epoxy DON in the 274 milk of the cows fed the Fusarium toxin contaminated wheat ranged between 0.1 and 0. varied between 4 % and 18 % and 82 % and 96 %, respectively. Based on the analysis of six 304 samples with and without incubation with β-glucuronidase, the degree of glucuronidation of 305 DON and de-epoxy DON in urine samples was found to be in the range of 21 % and 92 % and 306 86 % and 100 %. Daily DON and de-epoxy DON excretion in faeces of cows ranged between 307 89 and 1526 µg and 256 and 3978 µg in the mycotoxin period, whereas values in the control 308 period varied between 4 and 232 µg and 74 and 586 µg (Figure 6). Thereby, the portions of 309 DON and de-epoxy DON of DON intake were in the range of 0.4 up to 8.7 % and 1.2 up to 310 16.9 % when the Fusarium toxin contaminated wheat was fed and in the range of 0.2 up to 311 11.1 % and 3.1 up to 18.5 % feeding the control wheat. The total residues of DON plus de-312 epoxy DON in faeces ranged between 2 % and 18 % and between 8 % and 27 % of DON 313 intake in the mycotoxin and control period, respectively. DON and de-epoxy DON accounted 314 for from 5 % up to 44 % and from 56 % up to 95 % of the residues of both substances in 315 faeces when feeding the contaminated wheat and for 2 % up to 74 % and 27 % up to 98 % of 316 the residues of both substances in faeces when feeding the control wheat. 317 318 Discussion 319 Contradictory results have been obtained regarding the effects of DON on feed intake of 320 ruminants. For example, [START_REF] Noller | Effects of feeding Gibberella zeae-infected corn 520 on feed intake, body weight gain, and milk production of dairy cows[END_REF] found a slight decrease of daily feed intake with 321 a concomitant decrease of body weight when feeding Gibberella zeae infected corn in 322 complete mixed rations to lactating dairy cows. Similarly, [START_REF] Trenholm | Ingestion of vomitoxin (deoxynivalenol)-contaminated wheat by nonlactating 581 dairy cows[END_REF] noticed that 323 feed intake was slightly reduced when naturally contaminated grain (DON concentration up to 324 6.4 mg/kg) was added to the ration of nonlactating cows. In contrast, the feeding of diets 325 containing DON concentrations of 6 or 12 mg/kg [START_REF] Charmley | Influence of level of deoxynivalenol in 496 the diet of dairy cows on feed intake, milk production, and its composition[END_REF]) and up to 14 mg/kg 326 concentrate [START_REF] Ingalls | Influence of deoxynivalenol on feed consumption by dairy cows[END_REF] did not affect the feed intake. In the present study, the average 327 DON concentrations of the daily rations were 3.4 mg/kg and 0.3 mg/kg (at a reference DM 328 content of 88 %) in the mycotoxin and control period, so that the critical DON concentration 329 of 5 mg per kg of the total daily ration at a reference dry matter content of 88 % as issued for 330 dairy cows by the German Ministry of Consumer Protection, Food and Agriculture (BML 331 2000) was not exceeded. With regard to the feed intake, the change in feeding the control 332 wheat to feeding the Fusarium contaminated wheat did not show any distinctive features. 333 Due to the detoxifying ability of the rumen microflora, ruminants appear to have a higher 334 tolerance towards harmful substances such as DON. Several in vitro studies using bovine 335 rumen fluid have shown that the toxin is de-epoxidised to the less-toxic metabolite de-epoxy 336 DON [START_REF] King | Transformation of 517 deoxynivalenol (vomitoxin) by rumen microorganisms[END_REF][START_REF] Swanson | Metabolism of three trichothecene mycotoxins, T-2 toxin, diacetoxyscirpenol and 577 deoxynivalenol, by bovine rumen microorganisms[END_REF][START_REF] He | Microbial transformation of deoxynivalenol 511 (vomitoxin)[END_REF]). However, it is possible that a 337 modified toxin profile could alter the toxicity possibly responsible for the variable effects 338 observed when DON was fed to ruminants in practically relevant concentrations. So, the 339 present study was conducted to evaluate whether the feed intake, as an indicator for the feed 340 retention in the rumen, could alter the metabolisation and, therefore, the proportion of DON 341 and de-epoxy DON reaching the duodenum and eventually the milk. 342

In the present experiment, the proportion of DON and de-epoxy DON at the duodenum 343 relative to the DON intake varied between 12 % and 77 % when the Fusarium contaminated 344 wheat was fed (Table 3) and between 32 % and 153 % when the control wheat was fed. The 345 higher recovery of DON and de-epoxy DON at the duodenum in the control period is possibly 346 related to a higher variation coefficient of analytical results which is often found at lower 347 DON concentrations [START_REF] Whitaker | Sampling, 601 sample preparation, and analytical variability associated with testing wheat for 602 deoxynivalenol[END_REF]. In the mycotoxin period, the recovery of DON 348 and de-epoxy DON as a percentage of DON intake was relatively constant at a low level (12-349 23 %) at the lower feed/DON intakes, while an increased level of feed/DON intake resulted in 350 higher differences and recoveries (35-77 %). Furthermore, DON was almost completely 351 biotransformed to de-epoxy DON, whereby the de-epoxy DON portion of the duodenal flow 352 of both substances amounted up to 99 % in the mycotoxin period (Figure 1) and up to 97 % in 353 the control period. Likewise, [START_REF] Dänicke | On the effects of Fusarium toxins on the fermentation and 508 nutrient utilization in rumen of dairy cows[END_REF] who fed fistulated dairy cows with 354 Fusarium contaminated wheat (8.05 and 7.15 mg DON/kg wheat DM) over a 35 d period 355 found, that 89 % of the duodenal flow of both toxins were de-epoxy DON. Furthermore, 4-28 356 % of ingested DON were recovered at the duodenum in the form of DON and de-epoxy DON 357 at total daily DM intakes between 4 and 12 kg, which corresponds well with the recoveries of 358 12-35 % obtained at daily DM intakes between 6 and 12 kg in the present study. The slightly 359 higher recovery of 12-35 % compared to 4-28 % might possibly be caused by differences of 360 diet composition because, in contrast to the present experiment, the wheat portion of daily 361 ration amounted 50 % (on a DM basis) in the study of [START_REF] Dänicke | On the effects of Fusarium toxins on the fermentation and 508 nutrient utilization in rumen of dairy cows[END_REF], and rations were 362 completed by hay or grass silage. [START_REF] Dänicke | On the effects of Fusarium toxins on the fermentation and 508 nutrient utilization in rumen of dairy cows[END_REF] suggested that the low recovery of 363 DON as the sum of DON and de-epoxy DON at the duodenum would indicate either a 364 complete degradation of the toxin in the rumen or an absorption by mucosa of the rumen. This 365 presumption is supported by [START_REF] Prelusky | Plasma pharmacokinetics of the 531 mycotoxin deoxynivalenol following oral and intravenous administration to sheep[END_REF] who calculated from the areas under the 366 plasma-time concentration curves a mean pharmacokinetic bioavailability of 7.5 % following 367 administration of a single oral dose of 5 mg DON/kg b.w. and a single intravenous dose of 0.5 which indicates the liquid phase, was added in the rumen. Due to the intensive disappearance 370 rate of DON from the rumen which was two to three times faster than the marker the authors 371 also suggested a complete degradation and/or absorption of DON from the rumen. However, 372 conversion of DON to de-epoxy DON in the rumen was found to occur only up to 15 % 373 [START_REF] Prelusky | Plasma pharmacokinetics of the 531 mycotoxin deoxynivalenol following oral and intravenous administration to sheep[END_REF]. In the present study, and in the experiment by [START_REF] Dänicke | On the effects of Fusarium toxins on the fermentation and 508 nutrient utilization in rumen of dairy cows[END_REF], a 374 high proportion of the flow of DON and de-epoxy DON at the duodenum was the metabolite 375 de-epoxy DON (up to 99 % and 89 %). Due to the low variation of the proportion of de-epoxy 376 DON to DON of total DON plus de-epoxy DON flow at the duodenum between the different 377 DON/feed intakes in the current experiment, it can be concluded that DON is very rapidly 378 biotransformed to de-epoxy DON. Moreover, the lower biological recovery of DON plus de-379 epoxy DON at the lower DON/feed intakes could possibly indicates a complete degradation 380 of the toxin due to a higher ruminal retention time. Furthermore, de-epoxy DON was the 381 major component detected in the serum samples of cows fed the contaminated wheat in the 382 present study, while concentrations of DON were all below the indicated detection limit 383 (Figure 2). Moreover, the substance was only found as glucuronide conjugate, which implied 384 that the toxin will go directly to the liver where it undergoes an extensive glucuronidation in 385 the first-pass. In contrast, Sabater Vilar ( 2003) found a mean DON content of 32 µg/l using an 386 ELISA-method in blood serum of cows belonging to a Dutch farm. However, no data was 387 given by the authors regarding the applicability of the applied ELISA-method for 388 determination of DON in serum. The author supposed that the rumen was not able to degrade 389 the amount of DON to which the animals were exposed. The estimated individual daily DON 390 intake was 4.5 mg at an average daily DM intake of 20 kg, which is similar to the 391 concentration of 0.3 mg DON/kg DM control diet in the present study. However, in the 392 current experiment only trace amounts of de-epoxy DON were detected in the serum of the 393 cows fed the control wheat, whereas concentrations of DON were in all samples below the 394 detection limit. Accordingly, [START_REF] Prelusky | Metabolic-fate 537 and elimination in milk, urine and bile of deoxynivalenol following administration to 538 lactating sheep[END_REF] indicated that following an intravenous or 395 a large single oral dose of DON, only a small fraction was present in the blood plasma as de-396 epoxy DON [START_REF] Prelusky | Transmission 528 of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF][START_REF] Prelusky | Plasma pharmacokinetics of the 531 mycotoxin deoxynivalenol following oral and intravenous administration to sheep[END_REF][START_REF] Prelusky | Distribution of deoxynivalenol 540 in cerebral spinal fluid following administration to swine and sheep[END_REF]). However a 397 more prolonged oral exposure to a DON contaminated diet (330 and 880 mg/kg diet) for three 398 days resulted in higher amounts of de-epoxy DON in the plasma. The authors supposed an 399 efficient biotransformation of DON due to an extended exposure of DON to the rumen 400 microflora. A similar pattern of DON and de-epoxy DON was observed in the serum and in 401 the milk samples obtained from the present study. De-epoxy DON was detected as 402 predominant substance in the milk of the cows fed the contaminated wheat mainly occurring 4). However, trace amounts of DON were detected 404 beside the metabolite de-epoxy DON with the GC-MS method (Table 4), although, it needs to 405 be stressed that the detected DON levels were barely above the detection limit. In general, 406 knowledge about the carry over of DON and de-epoxy DON into the milk of dairy cows is 407 still deficient. While [START_REF] Charmley | Influence of level of deoxynivalenol in 496 the diet of dairy cows on feed intake, milk production, and its composition[END_REF] reported no transmission of DON and de-epoxy 408 DON in the milk of cows with a daily DON intake up to 104 mg (conjugated DON and de-409 epoxy DON were not analysed), [START_REF] Prelusky | Metabolic-fate 537 and elimination in milk, urine and bile of deoxynivalenol following administration to 538 lactating sheep[END_REF] found maximum total DON residue 410 levels of 222 ng/ml and 135 ng/ml, predominately as conjugated de-epoxy DON in milk of 411 sheep fed extremely high oral doses of 4.0 g and 1.5 g pure DON over a 72 hr period. Only 412 very low amounts of free and conjugated DON (< 4 ng/ml) were detected in the milk 413 following a large single oral dose of 1.7 mg DON/kg b.w. to lactating dairy cows (de-epoxy 414 DON was not analysed; [START_REF] Prelusky | Transmission 528 of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF]. Based on the levels of milk production, the 415 carry over rate of DON into the milk was almost negligible, only about 0.0001 % of the 416 administered dose. In the present experiment the total carry over rate of DON as DON and de-417 epoxy DON transmitted into milk ranged between 0.0001 and 0.0002 and 0.0004 and 0.0024 418 (GC-MS with enzyme incubation; Table 4). The results indicate a large difference in carry 419 over especially of de-epoxy DON between animals with lower and higher milk yields. For the 420 mycotoxin aflatoxin M 1 (AFM 1 , metabolite of aflatoxin B 1 ) it is supposed that there is a 421 passive concentration-dependent permeability of AFM 1 from blood to the alveolar cells of the 422 mammary gland [START_REF] Veldman | Carry-592 over of aflatoxin from cows food to milk[END_REF]). Possibly such a concentration-dependent permeability 423 exists for DON and de-epoxy DON, which could explain the significantly increasing total 424 carry over results of ingested DON as DON and de-epoxy DON in relation to the increased 425 milk production. Also, different permeabilities of the alveolar cell membranes might be 426 responsible for the variation in de-epoxy DON carry over. Furthermore, Veldman et al. 427 (1992) assumed that udder condition could also influence the carry over of AFM 1 because 428 udder infections can increase the permeability of the udder. Therefore, increased udder 429 permeability at the higher milk yields could also be a reason for the higher DON and de-430 epoxy DON contents in the milk. Further factors which could influence the DON and de-431 epoxy DON levels in milk might be equilibration between blood and other compartments, 432 DON biotransformation, entero-hepatic circulations, rates of urinary and faecal elimination 433 and rates of absorption from different areas of the gastrointestinal tract [START_REF] Prelusky | Transmission 528 of deoxynivalenol (vomitoxin) to milk following oral administration to dairy cows[END_REF]. 434

In the present study the daily DON and de-epoxy DON flows at the duodenum are 435 significantly linearly correlated with the daily DON and de-epoxy DON amounts in the milk 14 DON and de-epoxy DON residues were also analysed in urine and faeces samples. In the 438 current study de-epoxy DON was compared to DON the predominant substance in urine 439 (Figure 5), which was supposed to be the most important excretion route in a study of Cote et 440 al. (1986). The portion of de-epoxy DON of the total DON plus de-epoxy DON concentration 441 in urine amounted to 96 % when the Fusarium toxin contaminated wheat was fed. 442 Furthermore, DON, as well as de-epoxy DON residues in urine appeared mainly as 443 glucuronide conjugate (66 % and 98 %), which implied that glucuronic acid conjugation 444 appears to be an important metabolic pathway. However, although all animals were in a 445 steady feeding state, the urine concentrations of the present study should not be overvalued 446 because samples were collected spontaneously without consideration of water intake and the 447 kinetic behaviour of DON. Yet, the analyses show that DON and de-epoxy DON were also 448 detected in the urine of the cows fed the control wheat with lower concentrations of DON in 449 the diet. [START_REF] Prelusky | Excretion profiles 534 of the mycotoxin deoxynivalenol, following oral and intravenous administration to 535 lactating sheep[END_REF] found that only 7 % of DON and metabolites were eliminated 450 with the urine and 55 % up to 75 % of the DON dose appeared as unconjugated forms of 451 DON and de-epoxy DON in the faeces. However, the assumption that DON seems to 452 principally be excreted via the faeces can not be confirmed by the present data. Only from 2 453 % up to 18 % of consumed DON were excreted as DON plus de-epoxy DON in faeces when 454 the Fusarium contaminated wheat was fed. The method of DON administration possibly 455 influence the urinary and faecal excretion rates of DON and de-epoxy DON because, in 456 contrast to the current experiment, [START_REF] Prelusky | Excretion profiles 534 of the mycotoxin deoxynivalenol, following oral and intravenous administration to 535 lactating sheep[END_REF] administered a large single oral dose 457 of DON at a level of 5 mg/kg b.w. Furthermore, for the present experiment it has to be 458 stressed that the faeces samples were also taken spontaneously without consideration of the 459 kinetic behaviour of DON, however all animals were in a steady feeding state and the daily 460 amount of faeces from the cows was estimated using Cr 2 O 3 as marker. 461

  system equipped with individual feedbunks and had free access to water. 104

  Calculations and statistics 206 DM flow of the duodenal chyme and DM amount of the faeces were calculated by dividing 207 the amount of Cr dosed by the concentration of Cr of the duodenal digesta and faeces, 208 respectively. Concentrations of DON and de-epoxy DON in duodenal digesta and faeces were 209 calculated by multiplying mycotoxin concentration in duodenal digesta and faeces (on DM 210 basis) with the respective DM flow/DM amount.

  Mycotoxin flow at the duodenum 237

  epoxy DON in milk of cows fed the Fusarium toxin contaminated wheat ranged 269 between 1.6 and 2.7 µg/kg (HPLC-UV-method with β-glucuronidase incubation). 270

  total DON plus de-epoxy DON concentration in urine in the mycotoxin period 303

  concluded from this experiment that after the feeding of DON contaminated diets 464 (approximately 3.4 mg DON and 0.3 mg DON per kg complete ration, at a reference DM 465 content of 88 %, in the mycotoxin and control period) to dairy cows, DON is very rapidly 466 biotransformed to de-epoxy DON in the rumen due to the low variation of the portion of de-467 epoxy DON of the total flow of DON plus de-epoxy DON at the duodenum between the 468 different DON/feed intakes. Moreover, the lower biological recovery of DON plus de-epoxy 469 DON at the duodenum at lower DON/feed intakes could possibly indicate a complete 470 degradation of the toxin due to a higher ruminal retention time. Furthermore, excretion of -epoxy DON occurred through both urinary and faecal routes, with urinary 472 seeming to be the most important. Although very little amounts of were transmitted into the milk of mycotoxin fed animals, the increasing carry over rates of 474 DON and de-epoxy DON with an increasing milk yield need to be examined further. 475476Acknowledgement 477The assistance of the co-workers of the Institute of Animal Nutrition and the Experimental 478 Station of the Federal Agricultural Research Centre in Braunschweig, Germany in performing 479 the experiment and analyses as well as the support of this study by the H. Wilhelm 480 Schaumann Foundation (Hamburg, Germany) is gratefully acknowledged.

Figure 2 .(Figure 3 .Figure 4 .(Figure 5 .(Figure 6 .

 23456 Figure 2. Relationship between deoxynivalenol (DON) intake (mg/d) and de-epoxy DON residues in serum of dairy cows in the mycotoxin period Numbers over the symbols denote the individual cows 1 Measured by a high-performance liquid chromatography (HPLC) method

  EGMOND 2 , R. C. Braunschweig 38116, Germany, 2 Laboratory for Food and Residue Analyses (ARO), EU 10 Community Reference Laboratory for Residues (CRL), National Institute for Public Health 11 and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven, The Netherlands, 3 Institute of 12

	5
	6	SCHOTHORST 2 , A. A. JEKEL 2 , P. LEBZIEN 1 , M. SCHOLLENBERGER 3 , E. RAZZAZI-
	7	FAZELI 4 AND G. FLACHOWSKY 1
	8
	9	1 Institute of Animal Nutrition, Federal Agricultural Research Centre (FAL), Bundesallee 50,
	13	Animal Nutrition, Hohenheim University, Emil-Wolff-Str. 10, Stuttgart 70599, Germany,
	14	4 Department of Veterinary Public Health and Food Sciences/ Institute of Nutrition, University
	15	of Veterinary Medicine, Veterinarplatz 1, Vienna 1210, Austria
	16
	17

* Corresponding author: E-mail: karen.seeling@fal.de 18 19 20

Table 1 .

 1 Composition and mycotoxin concentrations of the concentrates and silages

	Concentrate 1)	Maize	Grass
	Contaminated	Control	silage	silage

Table 3 .

 3 Dry matter (DM) intake, deoxynivalenol (DON) intake, milk yield, daily flow of DON and de-epoxy DON and DON plus de-epoxy DON flow as percentage of DON intake at the duodenum of individual lactating cows in the mycotoxin period

	Cow	DM intake (kg/d)	DON intake (µg/d)	Milk yield (kg/d)	Flow of DON at the duodenum (µg/d)	Flow of de-epoxy DON (µg/d) at the duodenum	DON plus de-epoxy DON (% of DON intake)
	1	5.6 18800	-*	35	2104	12
	2	5.6 16570	-*	108	3650	23
	3	6.0 22620	-*	66	4059	18
	4	8.9 33840 9.6	225	4698	15
	5	11.4 38090 17.8	352	12170	33
	6	12.3 41040 15.4	185	5745	15
	7	12.6 47580 18.0	163	16539	35
	8	13.1 38780 19.7	526	12065	36
	9	15.3 42430 32.7	831	27659	67
	10	16.5 43510 29.1	1804	31505	77
	11	16.2 47900 23.7	362	24342	51
	12	17.8 47030 35.9	2193	32778	74
	13	19.9 75610 31.8	503	35064	47
	14	20.5 60760 42.7	663	26503	45

* Cows did not lactate

Table 4 .

 4 DON and de-epoxy DON concentrations (µg/kg) in the milk of individual lactating cows in the mycotoxin period determined with a HPLC-UV and a GC-MS method with and without β-Figure 1. Relationship between deoxynivalenol (DON) intake (mg/d) and percentage of DON and de-epoxy DON of the total DON plus de-epoxy DON flow at the duodenum as well as percentage of DON and de-epoxy DON of the total DON plus de-epoxy DON amount in the milk of dairy cows in the mycotoxin period Metabolite profile of DON and de-epoxy DON at the duodenum Metabolite profile of DON and de-epoxy DON in the milk Numbers over the symbols denote the individual cows

	glucuronidase incubation

1

Measured by a high-performance liquid chromatography (HPLC) method

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

(freeze-dried faeces and digesta) and 4 ng/ml (serum) and 10 ng/ml (urine). The recoveries for

Page 6 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Page 10 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

mg DON/kg b.w. to sheep. Furthermore, along with the oral dose, a cobalt-EDTA marker,

Page 12 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Page 13 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

(Figure4).

Page 14 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants

Page 15 of 28 http://mc.manuscriptcentral.com/tfac Email: fac@tandf.co.ukFood Additives and Contaminants


James William

MBBS Thomas Elston

Mirella Ling

Benjamin Jeffs

Kate Adams

Hiten Thaker

Peter Moss

Rolf Meigh

MD Gavin Barlow

Specialist James Elston
email: jameselston2003@yahoo.co.uk

Keywords: Staphylococcus aureus, serodiagnosis, anti-staphylolysin, anti-nuclease, antibody testing

The measurement of serum antibodies to S. aureus may be used in the diagnosis of deep seated infection, however, it remains unclear whether such testing is useful in clinical practice. We retrospectively identified results of serum anti-staphylolysin and anti-nuclease titres collected over a three year period. Serological testing was performed on 121 serum samples from 113 individuals between 1st September 2004 and 31st August 2007. Ninety (80%) individuals were managed as suspected or proven deep-seated infection. Serology results indicative of staphylococcal infection were returned in ten (9%) cases, including four where S. aureus was cultured. Serology results which did not indicate staphylococcal infection were returned in 90 (79.5%) cases including seven individuals with positive cultures for S. aureus. There was no evidence in any of the 113 cases that antimicrobial management was altered upon availability of test results. We conclude that staphylococcal serology tests are of no clear benefit in the management of suspected deep-seated infection.

AN EVALUATION OF THE USEFULNESS OF STAPHYLOCOCCUS AUREUS SERODIAGNOSIS IN CLINICAL PRACTICE.

James Elston, 1 Mirella Ling, 1 Benjamin Jeffs, 1 Kate Adams, 1 Hiten Thaker, 1 Peter Moss, 1 Rolf Meigh, 2 Gavin Barlow. 1 The measurement of serum antibodies to S. aureus has been used in the diagnosis of individuals with suspected deep seated infection, particularly when culture is difficult or unproductive. The Health Protection Agency (HPA), UK, currently offers testing to detect antibodies to alpha haemolysin (staphylolysin) and the nuclease enzyme in parallel. However, a lack of high specificity and sensitivity of the anti-staphylolysin test has been demonstrated repeatedly in occult S. aureus infections [START_REF] Verbrugh | Distinguishing complicated from uncomplicated bacteraemia caused by Staphylococcus aureus: the value of "new" and "old" serological tests[END_REF][START_REF] Ryding | Evaluation of seven different enzyme-linked immunosorbent assays for serodiagnosis of Staphylococcus aureus bacteraemia[END_REF][START_REF] Taylor | Serological tests in the differentiation of staphylococcal and tuberculous bone disease[END_REF], whilst there is little information on the usefulness of the anti-nuclease test. It is understood that serological tests, even in combination, are an imperfect marker of staphylococcal infection. It is unclear whether staphylococcal serological testing is useful in clinical practice.

We undertook a retrospective evaluation of the use of staphylococcal serodiagnosis in the Hull and East Yorkshire NHS Trust. Our objectives were to determine the extent of testing, to identify the clinical context in which tests were requested, to ascertain whether the results contributed towards patient management, and to inform the production of local guidelines.

We retrospectively identified results of serum anti-staphylolysin and anti-nuclease titres collected from individuals presenting to a 1500-bedded teaching hospital over a three year period. Information concerning the demographics of the individual, their relevant hospital and or outpatient attendances, past medical history, clinical diagnoses, microbiology, antimicrobial and medical management, and outcome was obtained from their clinical record and the trust pathology database. Test results were interpreted in accordance with HPA guidance [START_REF] Anon | Staphylococcus aureus serodiagnosis[END_REF]. A positive test was defined as an antistaphylolysin titre of ≥ 8 units/ml and/or an anti-nuclease titre of > 32 units/ml; an equivocal test (suggestive of staphylococcal infection), an antistaphylolysin titre of 4-6 units/ml and/or anti-nuclease titre of 32 units/ml; and a negative test (no evidence of Serology results indicative of staphylococcal infection were returned in ten (9%) cases, in four of these, S. aureus was cultured during the same hospital admission. Serology results suggestive of staphylococcal infection (equivocal) were returned in 13 (11.5%) cases; S. aureus was cultured from three of these during the hospital admission, (serum sample was taken within 48 hours of clinical presentation in all three cases) and an alternative pathogen was isolated in four cases. Serology results which did not indicate staphylococcal infection were returned in 90 (79.5%) cases (including seven individuals with positive cultures for S. aureus). Most individuals were treated with an antimicrobial regimen effective against S. aureus, unless an alternative pathogen was isolated (Figure 1). There was no evidence in any of the 113 cases that antimicrobial management was altered upon availability of test results.

In summary, most individuals tested were thought to have deep-seated infection although inappropriate testing in other clinical situations was observed. Serial serology was rarely performed. The small number of positive tests included cases with positive S. aureus culture; equivocal test results suggestive of S. aureus infection included cases where an alternative pathogen was isolated; and several "false negative" results in individuals with proven deep seated staphylococcal infection were noted. These results are consistent with the findings of previous studies [START_REF] Verbrugh | Distinguishing complicated from uncomplicated bacteraemia caused by Staphylococcus aureus: the value of "new" and "old" serological tests[END_REF][START_REF] Ryding | Evaluation of seven different enzyme-linked immunosorbent assays for serodiagnosis of Staphylococcus aureus bacteraemia[END_REF][START_REF] Taylor | Serological tests in the differentiation of staphylococcal and tuberculous bone disease[END_REF]. There was no evidence that test results influenced clinical decision making in any of the individuals tested. When alterations in antimicrobial management were observed and recorded it was noted that these decisions were made on the basis of clinical judgement supported by basic blood tests including inflammatory markers.

In clinical practice, the median time from sample collection to receipt of serological results of two weeks appears important. Antimicrobial therapy is usually initiated near to the time of the clinical presentation of an individual with deep seated infection, and response or non-response is likely to be evident clinically and or by serum markers of inflammation during the first 14 days of therapy. A shorter turnaround time for results is unlikely to be practical. The value of serological test results known to lack sensitivity and specificity in the context of a succeeding or failing antimicrobial regimen is questionable. Whilst serial antibody titres taken at intervals might potentially be useful in the diagnosis of selected cases of deep-seated infection, the 3 same limitations apply and it is therefore unlikely that they would affect clinical management. Further study is required to inform their use in clinical practice.

Given the known limitations of these tests and our observation that clinical management was not influenced by test results, we conclude that staphylococcal serology tests are of no clear benefit in the management of suspected deep-seated infection. We would suggest that testing should only be performed in highly selected cases where the degree and duration of suspected infection suggest the results might contribute.

Manuscript Click here to download Manuscript: manuscript 16022010.doc Click here to view linked References

		staphylococcal infection or repeat testing required), an antistaphylolysin titre of ≤ 2
	1	units/ml and anti-nuclease titre of ≤ 16 units/ml.
	2
	3 4 5	Serological testing was performed on 121 serum samples collected from 113 individuals between 1st September 2004 and 31st August 2007. 73 (65%) were male;
	6	their median age was 55 years (range 2-90). Testing was performed in both inpatient
	7 8 9 10	(81, 72%) and outpatient (32, 28%) hospital settings. Testing was requested on individuals managed by a variety of specialties including infectious diseases (78, 70%), orthopaedics (11, 10%), cardiology (3, 2.7%), neurosurgery (3, 2.7%) and the
	11	acute medical admitting teams (16, 14.5%). Ninety (80%) individuals were managed
	12	as suspected or proven deep-seated infection including cases of discitis, (20, 18%),
	13 14 15 16	prosthetic joint (15, 13%) and native (10, 9%) septic arthritis, osteomyelitis (18, 16%), soft tissue infection (15, 13%), infective endocarditis (10, 9%) and with bacteraemia of uncertain source (2, 1.7%). In the remainder (23, 20%) there was no
	17	evidence of infection. Test results were available a median of 14 days from the time
	18 19 20	of the serum sample collection. Repeat serology was performed on only six individuals.
	21
	22
	23
	24
	25
	26
	27
	28
	29
	30
	31
	32
	33
	34
	35
	36
	37
	38
	39
	40
	41
	42
	43
	44
	45
	46
	47
	48
	49
	50
	51
	52
	53
	54
	55
	56
	57
	58
	59
	60
	61
	62
	63 64	2
	65

*