Validation of the AnticFast® Beta-Lactams & Tetracyclines Combo Test Kit for Detection of Residues of Beta-Lactams (Penicillins and Cephalosporins) and Tetracyclines in Raw Cows' Milk: AOAC Performance Tested MethodSM 032403.

BACKGROUND: AnticFast® Beta-lactams & Tetracyclines Combo Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) and tetracycline antibiotic residues in raw commingled cows' milk. OBJECTIVE: The method performance was evaluated according to Commission Implementing Regulation 2021/808 and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines and submitted for AOAC Performance Tested Methods  SM certification. METHODS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit was evaluated for detection capability (CCß), selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU Maximum Residue Limits (MRLs) for ß-lactams and tetracyclines as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is specific for the detection of residues of ß-lactams and tetracyclines in milk and does not detect residues from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect (minimum a 95% detection) all residues of ß-lactams (penicillins and cephalosporins) and tetracyclines (parent drugs and their 4-epimers) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, with a 95% detection only above the MRL. No false positives were detected in 599 (305 blank farm and 301 tanker load) samples tested on both channels. Five real positives were detected and confirmed on the tetracycline channel for the blank farm milk, and two positives were detected and confirmed on the ß-lactam channel for the tanker samples. Robustness testing indicated that the detection in high protein raw cows' milk and heat-treated milk types (UHT, sterilized and reconstituted milk powder) may be slightly hampered. For substances with a detection capability well below the MRL this interference is not causing problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen for high fat raw cows' milk and for all other cows' milk types other than raw milk and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-lactams & Tetracyclines Combo Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam and tetracycline antibiotics. HIGHLIGHTS: The AnticFast Beta-lactams & Tetracycline Combo Test Kit is an easy, reliable, robust and highly specific test for screening of ß-lactam (penicillins and cephalosporins) and tetracycline antibiotic residues in milk with incubation at room temperature. In raw cows' milk, all tetracyclines are detected below 10 µg/kg.

Screening for antimicrobial residues in poultry eggs in Bangladesh using Charm II radio-receptor assay technique following validation.

The aim of the study was to screen for the presence of antimicrobial residues in poultry eggs from Bangladesh using the Charm II radio-receptor assay in the absence of expensive confirmatory instrumentation. This was based on cut-off values as set in the validation guidelines according to Commission Decision 2002/657/EC and Commission Implementing Regulation (EU) 2021/808. Fortified eggs spiked with fixed concentrations of doxycycline, erythromycin A, sulphamethazine, and benzylpenicillin were used to determine the cut-off values and detection capabilities (CCß). Other validation parameters included were applicability, ruggedness, and robustness. A total of 201 egg mix samples from native organic chicken, duck, and commercial farm-raised laying hens (both brown and white eggs) were tested and after analysis 13%, 10%, and 4.5% of the egg mix samples showed positive signals for sulphonamides, macrolides/lincosamides, and tetracyclines, respectively. Presence of multiple drug residues were also suspected in 11 out of 201 egg mix samples.

Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

BACKGROUND: AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. OBJECTIVE: The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. METHODS: The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of ß-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect all residues of ß-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam antibiotics. HIGHLIGHTS: AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

Honey bee exposure scenarios to selected residues through contaminated beeswax.

Twenty-two pesticides and veterinary drugs of which residues were detected in beeswax in Europe were selected according to different criteria. The risk to honey bee health posed by the presence of these residues in wax was assessed based on three exposure scenarios. The first one corresponds to the exposure of larvae following their close contact with wax constituting the cells in which they develop. The second one corresponds to the exposure of larvae following consumption of the larval food that was contaminated from contact with contaminated wax. The third one corresponds to the exposure of adult honey bees following wax chewing when building cells and based on a theoretical worst-case scenario (= intake of contaminants from wax). Following these three scenarios, maximum concentrations which should not be exceeded in beeswax in order to protect honey bee health were calculated for each selected substance. Based on these values, provisional action limits were proposed. Beeswax exceeding these limits should not be put on the market.

Primary validation of Charm II tests for the detection of antimicrobial residues in a range of aquaculture fish.

The study carried out a primary validation of Charm II tests for the detection of antimicrobial residues in aquaculture fish. The validation was performed according to European Commission Decision 2002/657/EC and the parameters determined included: detection capability, repeatability, reproducibility, specificity and robustness for the detection of antimicrobial residues in fish. Fish materials from different species including cat fish, trout, salmon, sea bass, tilapia, lingue and pangasius, were spiked with varying concentrations of selected antimicrobials including sulfonamides, ß-lactams, macrolides, tetracyclines and aminoglycosides to determine the detection capabilities and other validation parameters of the Charm II tests. Results of the validation showed that the detection capabilities for the tetracyclines ranged from 25 to 100 µg/kg, while the sulfonamides and aminoglycosides were detected at 25 µg/kg for all species under study. The detection capabilities for the beta-lactams ranged from 25 to 300 µg/kg; and was 100 µg/kg for the tested macrolides. Results of the study showed that there was no significant difference between counts for samples read immediately after addition of the scintillation liquid and those read 14 h after addition of the scintillation liquid, provided that there was good vortexing before analysis. There was also no significant difference between counts for the same samples analyzed in different runs under repeatability and reproducibility conditions at the same spiking concentrations for the different fish species analyzed. The relative standard deviation for both repeatability and reproducibility ranged from 1.2 to 15.1%. The Charm II tests were found to be 100% group specific, as none of the antimicrobials kits, gave false positive results when testing non-target antimicrobial drugs. Results of this study demonstrate the suitability of the Charm II technique as a rapid screening tool for detection of antimicrobial residues in a variety of fish species at maximum residue limits (MRL) established in the EU guidelines, with the exception of tilmicosin which was detected at 2 MRL. The results also prove the robustness, specificity, reliability and precision of the Charm II assay in the detection of various antimicrobial residuals in fish and its applicability for the rapid evaluation of the quality of aquaculture fish for safety and trade purposes.

Albendazole residues in goat's milk: Interferences in microbial inhibitor tests used to detect antibiotics in milk.

Albendazole (ABZ) residues in goat's milk and their effect on the response of microbial inhibitor tests used for screening antibiotics were evaluated. A total of 18 Murciano-Granadina goats were treated with ABZ and individually milked once a day over a 7-day period. ABZ quantification was performed by high performance liquid chromatography. The ABZ parent drug was not detected. The maximum concentration of its metabolites (ABZ sulfoxide, ABZ sulfone, and ABZ 2-aminosulfone) was reached on the 1st day post treatment (260.0 ± 70.1 µg/kg, 112.8 ± 28.7 µg/kg, 152.0 ± 23.6 µg/kg, respectively), decreasing to lower than the maximum residue limit (MRL, 100 µg/kg) on the 3rd day post treatment. Milk samples were also analyzed by microbial tests [Brilliant Black Reduction Test (BRT) MRL, Delvotest SP-NT MCS and Eclipse 100], and only one positive result was found for Delvotest SP-NT MCS and Eclipse 100. However, a high occurrence of positive outcomes was obtained for BRT MRL during 6 days post treatment, whereas ABZ residues were not detected from the 4th day post administration, suggesting that factors other than the antiparasitic agent might affect the microbial test response.

Residues in Beeswax: A Health Risk for the Consumer of Honey and Beeswax?

A scenario analysis in regard to the risk of chronic exposure of consumers to residues through the consumption of contaminated honey and beeswax was conducted. Twenty-two plant protection products and veterinary substances of which residues have already been detected in beeswax in Europe were selected. The potential chronic exposure was assessed by applying a worst-case scenario based on the addition of a "maximum" daily intake through the consumption of honey and beeswax to the theoretical maximum daily intake through other foodstuffs. For each residue, the total exposure was finally compared to the acceptable daily intake. It is concluded that the food consumption of honey and beeswax contaminated with these residues considered separately does not compromise the consumer's health, provided proposed action limits are met. In regard to residues of flumethrin in honey and in beeswax, "zero tolerance" should be applied.

Interferences on microbial inhibitor tests related to ivermectin treatment in lactating dairy goats.

This Research Communication reports interferences related to the administration of ivermectin in lactating dairy goats on the response of microbial tests for screening antibiotics in milk. Twenty-eight Murciano-Granadina goats, naturally infested with Sarcoptes scabiei var. caprae, were treated with a subcutaneous injection of ivermectin (200 µg/kg b.w.). To prevent re-infestation, a second dose was applied 7 d later. Individual milk samples were collected, daily, up to 15 d post-treatment. Milk samples were analysed by microbial inhibitor tests (BRT MRL, Delvotest SP-NT MCS and Eclipse 100) and ivermectin residues were quantified by HPLC. A large number of positive results were obtained for all microbial tests, especially on the first day after treatment (BRT MRL = 46·4%; Delvotest SP-NT MCS = 14·3%; and Eclipse 100 = 17·8%). However, the highest concentration of drug residues in milk (24·3 ng/ml) was detected on the tenth day after treatment, when positive outcomes were relatively lower (BRT MRL = 17·8%; Delvotest SP-NT MCS = 10·7%; and Eclipse 100 = 7·4%). Results herein suggest that factors related to the ivermectin treatment other than drug residues in milk, or alterations produced by the parasitic disease itself affecting the immune response of animals, could be the cause of false-positive results in microbial tests. It can be concluded that the application of ivermectin in dairy goats infested with sarcoptes mange during lactation produces persistent drug residues in milk, and could also cause false-positive results in microbial inhibitor tests for screening antibiotics.

Pesticides for apicultural and/or agricultural application found in Belgian honey bee wax combs.

In a Belgian pilot study honey bee wax combs from ten hives were analyzed on the presence of almost 300 organochlorine and organophosphorous compounds by LC-MS/MS and GC-MS/MS. Traces of 18 pesticides were found and not a single sample was free of residues. The number of residues found per sample ranged from 3 to 13, and the pesticides found could be categorized as (1) pesticides for solely apicultural (veterinary) application, (2) pesticides for solely agricultural (crop protection) application, (3) pesticides for mixed agricultural and apicultural (veterinary) application. The frequencies and quantities of some environmental pollutants bear us high concerns. Most alarming was the detection of lindane (gamma-HCH) and dichlorodiphenyltrichloroethane (including its breakdown product dichlorodiphenyldichloroethylene), two insecticides that are banned in Europe. The present comprehensive residue analysis, however, also reveals residues of pesticides never found in beeswax before, i.e. DEET, propargite and bromophos.

Validation of a lateral flow test (MRLAFMQ) for the detection of aflatoxin M1 at 50 ng l⁻¹ in raw commingled milk.

Aflatoxin M1 contamination in dairy products is a risk when feedstuff contaminated with aflatoxin B1 produced by moulds is consumed by milk-producing animals. Milk can be screened for aflatoxin M1 at the European Union maximum limit of 50 ng l⁻¹ by a lateral flow test, the MRLAFMQ (Aflatoxin M1) Test. The method takes 15 min with no milk dilution or a sample preparation step. The lateral flow assay was validated at the Technology and Food Science Unit of the Institute for Agricultural and Fisheries Research (ILVO-T&V) according to European Union guidelines using fortified raw milk samples. A detection capability of 50 ng l⁻¹ was demonstrated with a false negative rate lower than 2% at 50 ng l⁻¹ and a false positive rate of less than 0.3%. Quantitative readings had a mean bias of +2 to 6 ng l⁻¹ at 50 ng l⁻¹ with a standard deviation of 5-8 ng l⁻¹. Based on the validation results, the test could be considered appropriate for milk screening prior to milk unload at dairies.

Cyclic lipodepsipeptides produced by Pseudomonas spp. naturally present in raw milk induce inhibitory effects on microbiological inhibitor assays for antibiotic residue screening.

Two Pseudomonas strains, identified as closely related to Pseudomonas tolaasii, were isolated from milk of a farm with frequent false-positive Delvotest results for screening putative antibiotic residues in raw milk executed as part of the regulatory quality programme. Growth at 5 to 7°C of these isolates in milk resulted in high lipolysis and the production of bacterial inhibitors. The two main bacterial inhibitors have a molecular weight of 1168.7 and 1140.7 Da respectively, are heat-tolerant and inhibit Geobacillus stearothermophilus var. calidolactis, the test strain of most of the commercially available microbiological inhibitor tests for screening of antibiotic residues in milk. Furthermore, these bacterial inhibitors show antimicrobial activity against Staphylococcus aureus, Bacillus cereus and B. subtilis and also interfere negatively with yoghurt production. Following their isolation and purification with RP-HPLC, the inhibitors were identified by NMR analysis as cyclic lipodepsipeptides of the viscosin group. Our findings bring to light a new challenge for quality control in the dairy industry. By prolonging the refrigerated storage of raw milk, the keeping quality of milk is influenced by growth and metabolic activities of psychrotrophic bacteria such as pseudomonads. Besides an increased risk of possible spoilage of long shelf-life milk, the production at low temperature of natural bacterial inhibitors may also result in false-positive results for antibiotic residue screening tests based on microbial inhibitor assays thus leading to undue production loss.

Hydroxymethylfurfural: a possible emergent cause of honey bee mortality?

Hydroxymethylfurfural (HMF), a common product of hexose degradation occurring during the Maillard reaction and caramelization, has been found toxic for rats and mice. It could cause a potential health risk for humans due to its presence in many foods, sometimes exceeding 1 g/kg (in certain dried fruits and caramel products), although the latter still is controversial. HMF can also be consumed by honey bees through bad production batches of sugar syrups that are offered as winter feeding. In Belgium, abnormal losses of honey bee colonies were observed in colonies that were fed with syrup of inverted beet sugar containing high concentrations of HMF (up to 475 mg/kg). These losses suggest that HMF could be implicated in bee mortality, a topic that so far has received only little attention. This paper reviews the current knowledge of the presence of HMF in honey bee environment and possible consequences on bee mortality. Some lines of inquiry for further toxicological analysis are likewise proposed.

Antimicrobials in beekeeping.

The bee diseases American and European foulbrood and nosemosis can be treated with anti-infectious agents. However, in the EU and the USA the use of these agents in beekeeping is strictly regulated due to the lack of tolerance (e.g. Maximum Residue Limit) for residues of antibiotics and chemotherapeutics in honey. This article reviews the literature dealing with antimicrobials of interest in apiculture, stability of these antimicrobials in honey, and disposition of the antimicrobials in honeybee hives.

Validation of the Charm MRL-3 for fast screening of beta-lactam antibiotics in raw milk.

The biochemicals utilized in the Charm MRL beta-Lactam test (8 min test) were applied to faster flowing lateral components to create a new 3 min, one-step beta-lactam test called Charm MRL-3 (Charm Sciences Inc., Lawrence, MA). This new test was validated at T&V-ILVO according to Commission Decision 2002/657/EC. The following analytical parameters were checked: test specificity, detection capability, and test robustness (impact of deviation of the test protocol, and impact of the milk composition, batch differences of reagents). Further, the suitability of the Charm MRL-3 to screen heat-treated milk or milk from animal species other than the cow was also tested. Finally, the test was integrated in the monitoring of dairy samples to check the occurrence of false-negative or false-positive results, and the test was also included in a national ring trial and an international proficiency study. The results proved that the Charm MRL-3 is a fast, simple, and reliable cows' milk test that can be used at the farm level in order to prevent tanker milk contamination, or at the entrance of the dairy plant to screen tanker milk for the presence of beta-lactam antibiotics.

Transfer of sulfamethazine from contaminated beeswax to honey.

A liquid chromatographic tandem mass spectrometric method for the determination of sulfa drugs in beeswax was developed. When performing residue control on beeswax intended for the fabrication of wax foundations, residues of sulfonamides were found. A migration test was set up to study whether sulfonamide-containing beeswax could lead to the contamination of honey. The higher the concentration of sulfamethazine doped in the wax, the higher was the concentration of sulfamethazine found in the honey. The maximum transfer was 15.6, 56.9, and 29.5% of the initial amount spiked in the wax foundation. In a second experiment, the percentage of sulfamethazine migrating from medicated winter feed to beeswax in relation to the concentration in the syrup and the contact time was studied. The maximum transfer of sulfamethazine from medicated sucrose syrup to beeswax was 3.1%.

Validation of the tetrasensor honey test kit for the screening of tetracyclines in honey.

Regarding anti-infectious agents, no maximum residue limits are fixed for honey in the European legislation. Discussions are being conducted in order to set working limits at the European level; for example, for tetracyclines, 20 microg/kg was proposed. The Tetrasensor Honey test kit is a receptor-based assay using dipsticks for a rapid screening (30 min) of honey on the presence of tetracyclines. The test was validated according to Commission Decision 2002/657/EC. The test detects tetracycline, oxytetracycline, chlortetracycline, and doxycycline in honey in a specific and sensitive way. Depending on the type of tetracycline, detection capabilities (CCbeta) between 6 and 12 microg/kg were obtained (4-7 microg/kg for dried dipsticks). The test is rugged and participation with the test in an international ring trial gave compliant results. It can be concluded that the Tetrasensor Honey test kit is a simple and reliable test that can even be used at the production site.

Development of an indirect competitive ELISA for flumequine residues in raw milk using chicken egg yolk antibodies.

To detect flumequine in raw milk, an indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed. By carbodiimide conjugation, flumequine was conjugated to cationized bovine serum albumin (cBSA-flumequine) and to cationized ovalbumin (cOVA-flumequine). For the immunization of chickens, cBSA-flumequine was used, which allowed the isolation of specific chicken egg yolk immunoglobulins (IgY) for flumequine. As the coating antigen in the immunoassay, cOVA-flumequine was used. In the indirect competitive assay, standard flumequine was incubated together with the anti-flumequine antibodies. The antibody by which the lowest concentration of free flumequine that gives 50% inhibition of binding (IC50) was found in aqueous dilution was further tested for the applicability to detect flumequine in raw milk. An IC50 level in milk was reached that was about 5 times lower than in aqueous solution. So flumequine can be detected directly in raw milk at maximum residue level (50 microg/kg). No cross-reactivity was noticed with various related quinolones.