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J. Dairy Sci. 86:1947-1952
© American Dairy Science Association, 2003.

Evaluation of Screening Test for Detection of Antimicrobial Residues in Ewe Milk

M. P. Molina1, R. L. Althaus, S. Balasch, A. Torres, C. Peris and N. Fernandez

Departamento de Ciencia Animal, Departamento de Estadistica, Universidad Politécnica de Valéncia, Camino de Vera, 14, 46071 Valencia, Spain

Corresponding author:
M. P. Molina; e-mail:
pmolina{at}dca.upv.es.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The effects of preservatives (potassium dichromate and sodium azide), heat treatment (untreated and 82°C/10 min), and lactation stage upon the response of the microbial tests (BRT AiM and Delvotest) utilized for the detection of residues of antimicrobial substances in ewe milk were examined. Milk samples were collected from the morning milking of 50 Manchega ewes every 2 wk, from 15 d postpartum until the end of lactation. A total of 2322 samples were analyzed by BRT AiM with prediffusion and Delvotest microbial tests. The specificity of preservative-free milk samples without heat treatment was high (96.3% for BRT and 97.7% for Delvotest), with results improving for those samples thermally treated at 82°C/10 min (99.0% for BRT and 98.7% for Delvotest). Potassium dichromate produced a total inhibition of growth of Bacillus stearothermophilus with both methods. When acidiol was utilized, the specificity of the samples not treated thermally was lower compared with preservative-free milk samples for the BRT AiM (90.2%) and Delvotest (91.0%) methods, improving when the samples were thermally treated, both for BRT AiM (94.8%) and Delvotest (95.3%), given that the presence of the preservative increased the frequency of doubtful results. The lactation stage significantly affected the results of the methods, with a greater frequency of false-positive and doubtful cases toward the end of the cycle, especially in those samples preserved with acidiol. The greater selectivity in both methods was therefore obtained for preservative-free ewe milk samples with prior heat treatment taken at the beginning or in midlactation period.

Key Words: ewe milk • microbiological screening test • BRT AiM • Delvotest • inhibitors • antibiotic residues


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
The presence in raw milk of antimicrobial substances can have serious toxicological (Dewdney et al., 1991; Currie et al., 1998) and technical consequences (Mourot and Loussourorn, 1981; Brady et al., 1988). Moreover, the presence of chemical residues, particularly antibiotics, can delay (if not totally prevent) the bacteriological processes used in the manufacture of certain dairy products. It is therefore of fundamental importance to avoid the presence of these residues in milk in order to reduce problems during processing as well as to prevent their transmission to the consumers. Furthermore, in countries of the Mediterranean area (France, Greece, Italy, Portugal, Spain, etc.), ewe milk is used exclusively in the elaboration of yogurt and cheese, in many occasions starting from raw milk.

In recent years, different methods of analysis for the swift detection of residuals of inhibitors present in milk have been developed (IDF, 1991). One of the properties that characterizes these methods is specificity, defined as "the relation between the number of negative results and the total number of samples analyzed by means of a determined method, utilizing residue-free milk" (Sischo and Burns, 1993).

Microbiological inhibitor tests are widely utilized for the screening of inhibitors in milk, being quick, easy to use, and economical. Some of them using bacterial test strains such as Bacillus stearotermophilus var. calidolactis, for example the Brilliant Reduction Test (BRT) and Delvotest, are used in quality control laboratories throughout the European Union.

Several authors (Macaulay and Packard, 1981; Oliver et al., 1984; Seymour et al., 1988; Cullor, 1992; Halbert et al., 1996) have pointed out the problem of false-positive results in certain microbiological inhibitor tests. Among other possible causes, these results may be attributable to the presence of indigenous antimicrobial agents (e.g., lactoperoxidase, lactoferrin, and lysozyme enzymes) (Carlsson and Björck, 1987rck, 1989; Beukers, 1991; Schiffmann et al., 1992), high SCC (Carlsson and Björck, 1989; Tyler et al., 1992; Van Eenennaam, 1993), and FFA (Mäyrä-Mäkinen, 1990).

The microbiological inhibitor tests were developed to verify the quality of cow milk and have been evaluated in numerous research papers (Seymour et al., 1988; Sischo and Burns, 1993; Andrew et al., 1997). They have also been applied to goat milk (Anifantakis et al., 1988; Contreras et al., 1997; Zeng et al., 1996; 1998), although few studies have been carried out on ewe milk (Althaus et al., 1999).

The different chemical composition and the greater activity of some microbial growth-inhibiting enzymes such as lactoperoxidase (Althaus et al., 2001) in ewe milk compared to cow milk may be the cause of the different response rates of the microbiological inhibitor tests utilized in ovine.

For this reason, the aim of the present work is to assess the response of BRT AiM and Delvotest methods in antimicrobial-free ewe milk obtained in the course of lactation, in combination with the effect due to the addition of preservatives or pre-heat treatment of the samples, upon the specificity of the tests employed to evaluate the presence of false-positive results.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
Milk Samples and Chemical Analyses
In order to obtain antimicrobial-free milk samples during the complete lactation period, a flock of 50 Manchega ewes from the experimental farm at the Animal Science Department of the Universidad Politécnica de Valencia (Spain) was used. The lambs were separated from the ewes 24 h after parturition, ewes were milked twice a day (0800 and 1700 h) until the end of the lactation period (150 d). The flock was kept permanently in stalls; remained under similar environmental, handling, and feeding conditions throughout the whole experiment; and did not receive any drugs during the experiment.

Ewes were fed according to milk yield and rations were balanced to meet minimum recommendations (Boquier et al., 1988) throughout lactation, consisting of straw, alfalfa hay, orange pulp, beet root pulp, brewers grains, and a commercial concentrate for lactating ewes.

Milking routine for ewes did not include any udder preparation previous to teatcup attachment. Individual samples were collected 15, 30, 45, 60, 75, 90, 105, 120, and 135 d after lambing, during morning milking and placed in 100 ml sterile plastic containers and kept at 4°C until analysis.

Before analysis, the samples were divided into 6 aliquots in order to test the effects of addition of the preservative and thermal treatment of the sample prior to analysis. These were: preservative-free (untreated and heated at 82°C for 10 min); potassium dichromate (untreated and heated at 82°C for 10 min); acidiol (untreated and heated at 82°C for 10 min).

As preservatives a 4 µl/ml solution of acidiol (150 mg chloranphenicol, 1 ml ethanol, 3.5 g. sodium azide, 4.5 g sodium citrate 5H2O, in 100 ml of distilled water) or 10 µl/ml of 10% potassium dichromate solution were used.

Microbiological Screening Test
Milk samples were analyzed during the six-hour period after collection by BRT AiM with prediffusion (AIM-Analytik in Milch Produktions-und Vertriebs GmbH, Munchen, Germany) and Delvotest (DSM Food Specialities, Dairy Ingredients, Delft, The Netherlands) microbial tests. Antibiotic-free milk samples were taken as "negative control". As "positive control", milk samples added with 4 µg penicillin/kg were used. BRT AiM and Delvotest were carried out according to the instructions of the manufacturer. The indicator color changes of both methods were classified visually as either negative, doubtful, or positive.

Statistical Treatment of Data
To evaluate the effects of the preservative (P), heat treatment (HT) and stage of lactation (SL) on the responses of the microbiological inhibitor tests, the logistic regression model (Agresti, 1990) was used, by means of which the qualifications "positive" and "doubtful" were grouped. The logistic model used was:


where Pi = probability for "positive and doubtful" response/"negative" response; f(xi) = linear logit model.

The results were processed using the STEPWISE option from the LOGISTIC procedure of SAS (SAS, 1998). The statistical design for analyzing the effects of the preservative, heat treatment and lactation period upon the visual interpretation of the antibiotic residue screening test was carried out with the following model:


where Lijkl = the logit model; [Pijkl] = probability for the response category ("positive and doubtful"/"negative"); ß0 = intercept; ß1, ß2, ß3 = estimate parameter for the model; Pi = effect of preservative, in dummy variable (Z = 0: no preservative; Z = 1: preservative); HTj = effect of heat treatment, in dummy variable (X = 0: no heat treatment and X = 1: 82°C/10 min); SLk = effect of stage of lactation (n = 9); {varepsilon}ijkl = residual error.

The concordance coefficient (SAS, 1998) was applied as rank correlation between the observed responses and predicted probabilities.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 
BRT AiM Microbiological Inhibitor Test
Potassium dichromate produced a marked inhibitory effect in the microbiological inhibitor test, preventing the development of Bacillus stearothermophilus var. Calidolactis C-953, present as test microorganism in the BRT AiM microbiological inhibitor test. The frequency of false-positive results was 87.3% and of doubtful results 12.7%, with no negative results being obtained in either case.

For this reason, only the effect of the heat treatment on the frequencies of the responses of the BRT AiM method for preservative-free ewe milk samples and those preserved with acidiol appear in Table 1Go.


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  		Table 1. Effect of preservative and heat treatment of milk samples on BRT AiM with prediffusion test results.
 
The frequency of negative qualifications in preservative-free ewe milk samples not subjected to heat treatment was high (specificity 96.3%), as shown in Table 1Go. The prior heat treatment of preservative-free milk samples produced a reduction in the frequency of doubtful cases (from 2.1% to 1.0%) and the elimination of 1.6% of positive cases, improving the response of the method.

Preservation of the milk samples with acidiol, a widespread practice in milk quality laboratories, produces interference with the method, since the frequency of negative cases is reduced (specificity 90.2%) in comparison with the preservative-free samples (specificity 96.3%). In the samples preserved with acidiol, a reduction was also observed in the frequency of doubtful cases, from 9.3% to 4.7%, when the samples were heat treated.

The increase in specificity observed both in preservative-free milk samples and in the samples preserved with acidiol by both treatment may be due to the denaturalization of some of the natural inhibitors in milk that could interfere with the microbiological inhibitor tests (Mourot and Loussourorn, 1981; Carlsson and Björck, 1987rck, 1989; Shuren, 1995).

Study by means of the logistic regression model to predict the probability of obtaining "false-positive and doubtful" results when the BRT AiM with prediffusion test is used with ewe milk. As potassium dichromate possesses a marked antimicrobial effect and causes a high degree of interference with the method, the results obtained with this preservative were not included in the statistical analysis. The logistic regression indicated a significant effect for acidiol ({chi}2 = 18.9381; P < 0.0001), stage of lactation ({chi}2 = 17.4287; P < 0.0003), and heat treatment ({chi}2 = 9.4941; P < 0.0021).

The concordance of the logistic model was 70.7% and the frequency of "false-positive and doubtful" cases when the BRT AiM method was utilized with ewe milk samples can be calculated by means of the following equation:


([1])

where SL = effect of stage of lactation; {lambda}1 = 1.2110 for samples preserved with acidiol and {lambda}2 = -0.7933 for heat treatment samples.

In Figure 1Go are presented the percentages of "false-positive and doubtful" cases calculated using equation 1Go for the preservative-free samples and those with acidiol. In said figure it may be observed that the frequency of "false-positive and doubtful" cases increases with the course of lactation, being higher for those samples preserved with acidiol. This increase in the frequency of "false-positive and doubtful" cases could be attributable to the presence of indigenous antibacterial substances ( Carlsson and Björck, 1987; Beukers, 1991; Schiffmann et al., 1992) or high somatic cell count (Carlsson and Björck, 1989; Tyler et al., 1992; Van Eenennaam et al., 1993), given that the concentrations of some of the natural inhibitors increase as lactation progresses (Carlsson and Björck, 1989; IDF, 1991).



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  		Figure 1. False-positive and doubtful rate in ewe milk samples analyzed by BRT throughout lactation. ({blacktriangleup})Preservative-free and non-heated ewe milk samples, ({triangleup}) Preservative-free and heated (82°C/10 min) ewe milk samples, (•) Acidiol preservative and non-heated ewe milk samples, ({circ}) Acidiol preservative and heated (82°C/10 min) ewe milk samples.

 
As shown in Figure 1Go, the probability of obtaining "false positive and doubtful" results in preservative-free ewe milk samples and without heat treatment presented very low values, of less than 2% at the start of the cycle, whereas values increased to almost 7% towards the end of this period. Prior heat treatment of the samples produced a reduction in the frequency of cases of "false-positive and doubtful" results which, at the end of the lactation period, reached a maximum of 3%.

The interference of acidiol became more noticeable at the end of the lactation period, with 20% of "false-positive and doubtful" results, which could be reduced to 10% by heat treatment at 82°C/10 min. For this reason, it may be convenient to carry out further studies aimed at reducing the interference of this preservative, for instance by modification of the operative conditions of the test (incubation time), by reducing the concentrations of some of the components of acidiol (chloramphenicol or sodium azide) or by seeking other preservation alternatives for ewe milk samples.

Delvotest Microbiological Inhibitor Test
As observed in the BRT AiM test, the preservation of ewe milk samples with potassium dichromate produced total inhibition of growth development of B. stearothermophilus, var. calidolactis C-953, present in the Delvotest method, with 92.5% of false-positive cases, 8.5% doubtful and no negative results.

In Table 2Go, the effects of acidiol and heat treatment of ewe milk samples upon the qualifications of the Delvotest method are presented.


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  		Table 2. Effect of preservative and heat treatment of milk samples on Delvotest test results.
 
The prior heat treatment of ewe milk samples improved the results in preservative-free samples, producing a slight decrease in frequency of doubtful results (from 1.5% to 1.3%) and the elimination of the three false-positive cases, probably due to the denaturalization effect of heating on certain indigenous antibacterial substances present in ewe milk.

In preservative-free ewe milk samples and without heat treatment, the frequency of "false-positive and doubtful" results of this work (2.3%) is similar to that indicated by Andrew et al. (1977) in cow milk samples obtained on day 151 postpartum for Delvotest (3.05% false-positive rate). Moreover, Sischo and Burns (1993) and Charm and Zomer (1995), using the Delvotest test with cow milk samples, achieved results with specificity of 98% and 95%, implying false-positive percentages of 2% and 5%. Other authors (Macaulay and Packard, 1981; Seymour et al., 1988) have obtained higher rates of false-positive results in cow milk.

Zeng et al. (1996) reported 7% of false-positive cases when applying the Delvotest method with Alpina goat milk samples, whereas Contreras et al. (1997) determined a specificity with goat milk samples of 97% (3% false-positive rate). These values are similar to those obtained in this work from preservative-free ewe milk samples (97.6%), indicating that the Delvotest microbial test is equally suitable for the analysis of inhibitors in ewe milk.

In addition, in this case, acidiol produces a reduction in the frequency of negative cases (91.0%) compared with the preservative-free ewe milk samples (97.7%). This fact confirms the need to adapt the operative conditions of the method when using ewe milk samples preserved with acidiol, or to investigate the use of another preservative that causes less interference with the Delvotest microbial test.

The heat treatment of the samples preserved with acidiol also produces a reduction in doubtful cases, which is more evident in cases where no preservative is used, as the frequency of doubtful results diminished from 8.5% to 4.7%, with the disappearance of the two false-positive results.

In order to consider the frequency of "false-positive and doubtful" cases when the Delvotest is utilized with ewe milk, the logistic regression model was used.

As with the BRT AiM method, the effects of acidiol ({chi}2 = 19.7226; P < 0.0001), stage of lactation ({chi}2 = 20.5770; P < 0.0001) and heat treatment ({chi}2 = 7.0952; P < 0.0077) were significant. The concordance percentage of the logistic model was 73.6%.

The equation that allows consideration of the frequency of "false-positive and doubtful" results when the Delvotest method is utilized with ewe milk samples is as follows:


([2])

where SL = effect of stage of lactation; {lambda}1 = 1.3719 for samples preserved with acidiol and {lambda}2 = -0.7242 for samples subjected to heat treatment (82°C/10 min).

In Figure 2Go, the relative frequencies of "false-positive and doubtful" cases calculated by means of equation 2Go are represented. The responses of the Delvotest method for ewe milk samples preserved with potassium dichromate have not been reported, due to its powerful antibacterial action. In said Figure, the interference of acidiol with the Delvotest microbial test and the increase in frequency of "false-positive and doubtful" cases as the lactation stage progresses can be observed.



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  		Figure 2. False-positive and doubtful rate in ewe milk samples analyzed by Delvotest throughout lactation. ({blacktriangleup}) Preservative-free and non-heated ewe milk samples, ({triangleup}) Preservative-free and heated (82°C/10 min) ewe milk samples, (•) Acidiol preservative and non-heated ewe milk sample, ({circ}) Acidiol preservative and heated (82°C/10 min) ewe milk samples.

 
The estimated percentages of "false-positive and doubtful" cases when the Delvotest method is applied with preservative-free ewe milk samples are low at the beginning of lactation (1%), reaching 6% at the end of this period. This may be due to the milk composition (much richer in fat and protein), the greater number of somatic cells and certain natural inhibitors associated with them at the end of lactation. Heat treatment reduces the "false-positive and doubtful" to values of less than 1% at the start of lactation and approximately 3% at the end. When the sample is heated, part of the natural inhibitors may be destroyed and there is therefore less interference with the test response.

In the case of samples with acidiol, there appears to be a synergetic effect between the false-positive and doubtful results due to the effect of the acidiol, which contains chloramphenicol in the formula, and the composition of ewe milk at the end of lactation. The values calculated by logistic model for acidiol ewe milk samples were high at the end of lactation, reaching values of approximately 20% at the end of the cycle in samples not thermally treated and of 10% for heat-treated samples (82°C/10 min). As in the case of the BRT AiM method, further studies leading to diminution of preservative interference are required.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
ACKNOWLEDGEMENTS
 REFERENCES
 
The specificity of preservative-free ewe milk samples and those without heat treatment was high for both methods, increasing when the milk samples were heated to 82°C/10 min. When acidiol is used as preservative the specificity is lower, especially in samples without heat treatment whereas Potassium dichromate produces total interference with the microbiological inhibitor tests.

It would therefore seem advisable to carry out heat treatment of ewe milk samples before applying the BRT AiM with prediffusion and Delvotest microbial tests. However, from a practical and cost-effective viewpoint, it may be recommendable to analyze all samples first without prior treatment. The positive and doubtful outcomes from said analysis would then require confirmation by sample heat treatment.

The highest frequencies of false-positive and doubtful results are present at the end of the lactation period, which in the case of acidiol being used as preservative could lead to an excessively high incidence of doubtful interpretations.

Given that acidiol is a preservative commonly used in milk quality laboratories, further studies aimed at the diminution of the interference of acidiol with these microbiological inhibitor tests are recommended, modifying operative conditions (incubation time) or the concentrations of some of their components, although it would be desirable to improve the conditions of sample collection and avoid the use of preservatives in these analyses.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
REFERENCES
 
This work forms part of the project ALI98-0363, financed by the Comisión Interministerial de Ciencia y Tecnología (Madrid, Spain). The authors are grateful to AIM-Analytik in Milch Produktions-und Vertriebs GmbH (Munchen, Germany) and DSM Food Specialties, Dairy Ingredients (Delft, The Netherlands) for their support. English text revised by Mr. Neil Macowan.


   FOOTNOTES
 
1 Current address: Departamento de Ciencias Básicas. Facultad de Ciencias Veterinarias. Universidad Nacional del Litoral. R.P.L. Kreder 2805. 3080 Esperanza, Argentina. Back

Received for publication February 16, 2002. Accepted for publication February 3, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 ACKNOWLEDGEMENTS
 REFERENCES
 


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